Large-scale biomolecular analysis with ssequence tags

ABSTRACT

The invention is directed to sequence-based profiling of populations of nucleic acids by multiplex amplification and attachment of one or more sequence tags to target nucleic acids anchor copies thereof followed by high-throughput sequencing of the amplification product. In some embodiments, the invention includes successive steps of primer extension, removal of unextended primers and addition of new primers either for amplification (for example by PRC) or for additional primer extension. Some embodiments of the invention are directed to minimal residual disease (MRD) analysis of patients being treated for cancer. Sequence tags incorporated into sequence reads provide an efficient means for determining clonotypes and at the same time provide a convenient means for detecting carry-over contamination from other samples of the same patient or from samples of a different patient which were tested in the same laboratory.

This application claims priority from U.S. provisional application Ser.No. 61/841,878 filed 1 Jul. 2013 and Ser. No. 62/001,580 filed 21 May2014, both of which are incorporated herein by reference in theirentireties.

BACKGROUND OF THE INVENTION

Large-scale DNA sequencing in diagnostic and prognostic applications hasexpanded rapidly as its speed and convenience has increased and itsper-base cost has decreased, e.g. Ding et al. Nature, 481(73.82):506-510 (2012); Chiu et al. Brit. Med. J., 342: c7401 (2011); Ku et al,Annals of Neurology, 71(1)5-14 (2012); and the like. In particular,profiles of nucleic acids encoding immune molecules, such as T cell or Bcell receptors, or their components, contain a wealth of information onthe state of health or disease of an organism, so that the use of suchprofiles as diagnostic or prognostic indicators has been proposed for awide variety of conditions. e.g. Faham and Willis, U.S. Pat. Nos.8,236,503 and 8,628,927; Freeman et al, Genome Research, 19: 1817-1824(2009); Han et al, J. Immunol, 182. (1001):42.6 (2009); Boyd et al, Sci.Transl. Med., 1(12): 12ra23 (2009); He et al. Oncotarget (Mar. 8, 2011).

For example, patients treated for many cancers often retain a minimalresidual disease (MRD) related to the cancer. That is even though apatient may have by clinical measures a complete remission of thedisease in response to treatment, a small fraction of the cancer cellsmay remain that have, for one reason or another, escaped destruction.The type and site of this residual population is an important prognosticfactor for the patient's continued treatment, e.g. Campana. Hematol,Oncol. Clin. North Am., 23(5): 1083-1098 (2009); Buccisano et al, Blood,119(2): 332-341 (2012). Consequently, several techniques for assessingthis population have been developed, including techniques based on flowcytometry, in situ hybridization, cytogenerics, amplification of nucleicacid markers, and the like, e.g. Buccisano et al, Current Opinion inOncology, 21: 582-588 (2009); van Dongen et al, Leukemia, 17(12):2257-2317 (2003); and the like. The amplification of recombined nucleicacids encoding segments of immune receptors (i.e. clonotypes) from Tcells and/or B cells have been particularly useful in assessing MRD inleukemias and lymphomas, because such clonotypes typically have uniquesequences which may serve as molecular tags for their associated cancercells. Such measurements are usually made by amplifying and sequencing,nucleic acids encoding a single receptor chain, in part, because suchamplifications are highly multiplexed and are difficult to develop. Asthe scale of multiplexing increases, several problems are encountered,including increased probability of spurious amplifications due tomis-hybridizations, primer-dimer formation, variable rates ofamplification leading to biased sequence representation, and the like,e.g., Elnifro et al, Clinical Microbiology Reviews, 13(4): 559-570(2000). Furthermore, the similarity of the target sequences and theincorporation of sequence tags into amplified sequences, either forsequence analysis, sample tracking, contamination detection, or thelike, can exacerbate the above difficulties associated with large-scaleamplifications. These challenges have prevented the development oflarge-scale one-reaction amplifications of multiple immune receptorchains, which would he highly beneficial for reducing the number ofseparate assays required for measuring nucleic acid sequences correlatedwith a minimal disease.

In view of the foregoing, it would be highly advantageous if moreefficient methods were available for assessing selected nucleic acids ina single reaction, such as exons of cancer genes or clonotypes encodingsets of immune receptor chains.

SUMMARY OF THE INVENTION

The present invention is directed to methods of large-scaleamplification in a single reaction, particularly by a polymerase chainreaction (PCR), of a population of target polynucleotides, such asrecombined nucleic acids encoding immune receptor chains, followed bytheir identification using large-scale DNA sequencing. The inventionincludes the application of the foregoing methods for monitoring minimalresidual disease of a cancer. The invention is exemplified in a numberof implementations and applications, some of which are summarized belowand throughout the specification.

In some embodiments, the invention is directed to methods of generatingprofiles of nucleic acids that encode a population of biomolecules ofinterest, such as immune receptor molecules. In one, aspect, methods ofthe invention comprise attaching sequence tags to a selected populationof nucleic acids in a sample to form tag-nucleic acid conjugates,amplifying, the tag-nucleic acid conjugates, and sequencing amplifiedtag-nucleic acid conjugates to provide sequence reads each comprisingboth a tag sequence and a nucleic acid sequence, for which a profile ofthe nucleic acids is generated. In some embodiments, attaching sequencetags is enabled by one or more successive steps of primer extension andpruner removal, after which the resulting product may be furtheramplified without bias by common forward and reverse primers.

In some embodiments, the invention is directed to methods for detectingand measuring contamination, such as carry-over contamination, in asample from material originating from a (baron sample, in oneembodiment, such method for detecting contamination in an individualbeing monitored for a minimal residual disease may comprise thefollowing steps: (a) obtaining from an individual a tissue sample; (b)attaching sequence tags to cancer gene molecules or recombined nucleicacids to form tag-nucleic acid conjugates, wherein at least one nucleicadd or copies thereof have different sequence tags attached and whereinthe cancer gene molecules are characteristic of a cancer of theindividual; (c) amplifying the tag-nucleic acid conjugates; (d)sequencing a sample of the tag-nucleic acid conjugates to providesequence reads having error rates and comprising a tag sequence and acancer gene sequence or recombined nucleic acid sequence; (e) comparingtag sequences to separately determined tag sequences from other tissuesamples; and (f) determining the presence, absence anchor level ofcontamination by the identity of one or more tag sequences with anyseparately determined tag sequences from other tissue samples.

In another aspect, the invention is directed to a method as describedabove for generating clonotype profiles based on at least two chains ofa B cell receptor, which method comprises amplifying in a singlereaction target nucleic acids encoding two or more chains of a B cellmentor. In another aspect, such methods are employed to monitor minimalresidual disease in a B cell cancer.

In another aspect, the invention is directed to a method as describedabove generating clonotype profiles based on at least two chains of a Tcell receptor, which method comprises amplifying in a single reactiontarget nucleic acids encoding two or more chains of a T cell receptor.In another aspect, such methods are employed to monitor minimal residualdisease in a T cell cancer.

These above-characterized aspects, as well as other aspects, of thepresent invention are exemplified in a number of illustratedimplementations and applications, some of which are shown in the figuresand characterized in the claims section that follows. However, the abovesummary is not intended to describe each illustrated embodiment or everyimplementation of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention is obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIGS. 1A through 1C illustrate diagrammatically various embodiments ofthe invention.

FIG. 1D illustrates a method of generating (with or without sequencetags) templates of recombined nucleic acids having a predeterminedlength.

FIGS. 2A through 2G illustrate various methods for attaching uniquesequence tags to substantially every target sequence in a sample.

FIGS. 3A and 3B illustrate diagrammatically an aspect of the inventionfor generating clonotype profiles from nucleic acid sequences encodingIgH chains.

FIG. 4A illustrates the use of sequence tags for determining clonotypesequences from sequence reads.

FIG. 4B illustrates the use of sequence tags in embodiments wheremultiple different sequence tags are attached to the same targetpolynucleotide or copies thereof.

FIG. 5A illustrates concept of clonotypes in sequence space anddistances between closely related clonotypes.

FIG. 5B is a flow chart illustrating one embodiment of a method fordistinguishing genuinely different clonotypes from clonotypes thatdiffer solely by sequencing errors (which should be coalesced).

FIG. 5C illustrates the form of a numerical function used in oneembodiment for determining whether or not to coalesce relatedclonotypes.

FIGS. 5D and 5E illustrate the use of sequence trees in a method ofcoalescing sequence reads.

DETAILED DESCRIPTION OF THE INVENTION

The practice or the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of molecular biology(including recombinant techniques), bioinformatics, cell biology, andbiochemistry, which are within the skill of the art. Such conventionaltechniques include, but are not limited to, sampling and analysis ofblood cells, nucleic, acid sequencing and analysis, and the like.Specific illustrations of suitable technique can be had by reference tothe example herein below. However, other equivalent conventionalprocedures can, of course, also be used. Such conventional techniquesand descriptions can be found in standard laboratory manuals such asGenome Analysis: A Laboratory manual Series (Vols. I-IV); PRC Primer: ALaboratory Manual; and Molecular Cloning: A Laboratory Manual (all fromCold Spring Harbor Laboratory Press) and the like.

In one aspect the invention is directed to methods for producingclonotype profiles of multiple immune receptor chains by huge-scalemultiples amplification of nucleic acids encoding such chains followedby high-throughput sequencing of the amplification product, or amplicon.In some embodiments, the invention overcomes common drawbacks ofmultiplex amplification by including successive steps of primerextension, removal of unextended, or unincorporated, primers andaddition of new primers either for amplification (for example by PCR) orfor additional primer extensions. Such steps also enable the use ofsequence tags which otherwise would contribute to nonspecific orspurious amplifications. In another aspect, sequence tags are employedin embodiments with clinical applications particularly minimal residualdisease (MRD) analysis, for example, of samples from a patient beingtreated for a cancer. Sequence tags incorporated into sequence readsprovide an efficient means for determining clonotypes and at the sametime provide a convenient means for detecting carry-over contaminationby detecting the presence or absence of sequence tags from previousassays, either front samples of the same patient or from samples of adifferent: patient which were tested in the same laboratory. Ofparticular interest are methods for generating sequence-based clonotypeprofiles of recombined nucleic acids encoding a plurality of B cellreceptor (BCR) chains by using a single amplification reaction followedby high throughput next-generation sequencing. Also of particularinterest are methods for generating sequence-based clonotype profiles ofrecombined nucleic acids encoding a plurality of T cell receptor (TCR)chains by using a single amplification reaction followed by highthroughput next generation sequencing. Methods of the invention may alsobe applied other large-scale amplification and sequencing of other setsof nucleic acids of interest, including, for example, sets of exons ofcancer genes. In these aspects, sequence tags permit both monitoring ofcarry over contamination and more sensitive determination of nucleotidesequences of target polynucleotides in view of error-prone sequencingmethodologies. Also in these aspects, a set of sequence tags (asdiscussed more fully below) is typically much larger than the number oftarget polynucleotides in a sample and the sequence difference amongsequence tags attached to target polynucleotides is large enough so thateffectively a sequence of one tag could not be transformed into anotherby sequencing error.

One embodiment of the invention is illustrated in FIG. 1A. In a reactionmixture, primers (22) from a first set (each primer of the first sethaving receptor-specific portion (16) and 5′-non-complementary portion(15) comprising a first primer binding site) anneal to one end of targetpolynucleotides (10) (after melting target polynucleotide (10)) andprimers (24) from as second set (each primer of the second set havingreceptor-specific portion (20) and 5′-non-complementary portioncomprising sequence tag (14) and second primer binding site (12)) annealto another end of target polynucleotides (10). In some embodiments, asnoted below, non-complementary portion (15) of primer (22) may alsocomprise a sequence tag. In some circumstance, two shorter sequence tagsmay be more advantageous than a single longer sequence tag of equivalentdiversity. Thus, for example, two 8-mer random-nucleotide sequence tagsmay be less likely to cause spurious priming, primer-dimers, and thelike, than a single 16-mer random nucleotide sequence tag. Targetpolynucleotides (10) are typically somatically recombined nucleic acidsfrom T cells or B cells which encoded chains or portions of chains of Tcell receptors (TCRs) or B cell receptors (e.g., portions of IgH chainsor IgK chains). Thus, in some embodiments, the receptor-specificportions of primers (22) and (24) may be specific for V region,sequences and J region sequences, respectively, or in other embodiments,vice versa.

In some embodiments, target polynucleotides (10) may comprise complexmixtures of nucleic acids whose sequence profiles are desired, includingbut not limited to, recombined nucleic acids encoding portions of immunereceptor molecules, 16S rDNAs of microbial communities, metagenomicamplifications of genes encoding proteins of industrial or medicalimportance (such as, enzymes), human or animal genes and/or exonsrelated to specific diseases, such as cancer, infectious disease, or thelike. In embodiments relating to recombined nucleic acids encodingimmune receptors, usually at least portions of a V, D or J region arepresent between the two binding locations of the first and second setsof primers. In some embodiments, between the two binding locations ofthe first and second sets of primers there is at least a portion of aVDJ rearrangement of IgH, a DJ rearrangement of IgH, at VJ rearrangementof IgK, a VJ rearrangement of IgL, a VDJ rearrangement of TCR β, a DJrearrangement of TCR β, a VJ rearrangement of TCR α, a VJ rearrangementof γ, a VDJ rearrangement of TCR δ, or a VD rearrangement of TCR δ. Insome embodiments, between the two binding locations of the first andsecond sets of primers there is at least a portion of a VDJrearrangement of IgH, as DJ rearrangement of IgH, a VJ rearrangement ofIgK, or is VJ rearrangement of IgL. In some embodiments, between the twobinding locations of the first and second sets of primers there is atleast a portion of a VDJ rearrangement of TCR β, as DJ rearrangement ofTCR β, a VJ rearrangement of TCR α, a VJ rearrangement of TCR γ, a VDJrearrangement of TCR δ, or a VD rearrangement of TCR δ. In still otherembodiments, between the two binding locations of the first and secondsets of primers there is at least as portion of a VDJ rearrangement ofIgH, a DJ rearrangement of IgH, and a VJ rearrangement of IgK. And inother embodiments, between the two binding locations of the first andsecond sets of primers there is at least a portion of a VDJrearrangement of TCR β, a VJ rearrangement of TCR γ, and a VDJrearrangement of TCR δ or a VD rearrangement of TCR δ, In someembodiments, at least a portion of a VDJ rearrangement comprises thecomplete D or NDN portion and parts of the V and J segments sufficientfor their identification. In some embodiments, at least a portion of aVDJ rearrangement comprises at least a 50 nucleotide segment comprisingthe complete D or NDN portion and parts of the V and J segments. In someembodiments, at least a portion of a VDJ rearrangement comprises atleast a 70 nucleotide segment comprising the complete D or NDN portionand pans of the V and J segments.

In some embodiments, a first set comprises one or more primers that areeach specific for a J segment or a C segment. Primers from such a firstset are annealed to their target sequences and are extended, after whichnon-extended primers of the first set are removed. Primers from a secondset that are each specific for a V segment are annealed to their targetsequences and are extended. In other embodiments, a first set comprisesprimers that are each specific for a V segment and primers of such firstset are annealed to their target sequences, and are extended, afterwhich non-extended primers of the first set are removed, printers of asecond set that are each specific for a J segment or a C segment areannealed to their target sequences and are extended. In alternatives ofboth of these embodiments, first and second sets may each contain aplurality of primers and each primer may be specific for a differentimmune receptor segment.

Returning to FIG. 1A, in some embodiments, primers of the first andsecond sets are extended (5) by carrying out in alternative embodiments,1-10, or 2-10, or 3-10, or 4-10, or 5-10 cycles of melting, annealingand extension, after which non-extended primers are removed from thereaction mixture using conventional techniques. In other embodiments,primers of the first and second sets are extended (5) by carrying out inalternative embodiments, 2-5, or 3-5, or 4-5 cycles of melting,annealing and extension, after which non-extended primers are removedfrom the reaction mixture using conventional techniques. In stillanother embodiment, primers of the first and second sets are extended bycarrying out two cycles of melting, annealing and extending. Forexample, non-extended primers may be removed by exonuclease digestion,hybridization to complementary sequences on magnetic beads, sizeexclusion chromatography, commercially available spin columns (e.g.Qiagen QIAquick PCR Purification Kit), or the like. In one embodiment,unextended, or unincorporated, primers are removed, for example, bydigestion with an exonuclease I. Double stranded DNAs (18), which areproducts of extensions (5), have common first and second printer bindingsites at each end, to which (in some embodiments) forward and reverseprinters, with complementary sequences (6 and 11), may be added forlater generation of clusters by bridge PCR. In some embodiments, doablestranded DNA also has sequence tag (19) and forward or reverse primermay include sample tag (2) for identifying or tracking or associatingDNA (18) with a sample or patient. In some embodiments, sequence tag(19) is substantially unique for each different recombined nucleic acidin a sample. As explained more fully below, sequence tag (19) may beused for coalescing sequence reads into clonotypes well as used fordetecting and tracking sample contamination. Forward and reverse primersmay also include primer binding sites (4) and (8) for implementing (13)bridge PCR for certain sequencing protocols, e.g. on a Genome Analyzer(Illumina, San Diego)(17). In other embodiments, in which more than oneextensions are carried out with sequence tag-containing primers, eachdifferent recombined nucleic acid in a sample may have copies withdifferent sequence tags attached; thus, for example, if four separatecycles of melting, annealing and extension are carried out on targetpolynucleotides in accordance with the embodiment of FIG. 1A, and if thesample contains recombined nucleic acid, S_(t), then at the completionof amplification (13) with common primers, the copies of S₁ will have upto four different sequence tags, Therefore, sequence reads of S₃ willhave up to four different sequence tags. As explained more fully below,in such embodiments, clonotypes may be determined by a combination ofaligning sequence tags and coalescing sequence reads within each subsetdefined by a common sequence tag.

In another embodiment, at least two extensions and two steps to removeunincorporated primers are implemented prior to PCR with common primers.As illustrated in FIG. 1B, primers (101) are annealed to one end oftarget polynucleotides (100), such as recombined nucleic acids encodingimmune receptor chains, and extended, e.g. with a DNA polymerase.Primers (101) may each include receptor-specific portion (103) and 5′non complementary portion (105) which, in turn, comprises sequence tag(104) and first primer binding site (102). After extension and removalof unincorporated printers (130), as described above, to first extensionproduct (109) in the reaction mixture is added (a) primers (125),wherein each primer comprises receptor-specific portion (106) and5′-non-complementary portion (115) (that contains a primer bindingsite), and (b) printers (127), comprising portion (108) specific forfirst printer binding site (102) and 5′ non-complementary portion (117).After primers (125) and (127) anneal to their printer binding sites,they are extended (107) to form second extension product (118), afterwhich non-extended printers are removed. To second extension product(118) common forward printers (112) and reverse primers (110) are addedand a PCR is implemented (111), after which the resulting amplicon issequenced (120). As above with the embodiment of FIG. 1A, whenever morethan one extension step is performed in the presence of sequencetag-containing printers (such as (101)), copies of the same targetpolynucleotide (100) may be labeled with a plurality of differentsequence tags.

FIG. 1C illustrates another embodiment with V, D and J regions shownexplicitly. In a reaction mixture under primer annealing conditions, torecombined nucleic acids (1200) encoding immune receptors, such as TCRs,primers (1212), a first Set of primers specific to V region (1226), isadded. Each primer of the first set (1212) includes a receptor-specificportion and a 5′-non-complementary portion which, in turn, comprisesoptionally a sequence tag and a first printer binding site (e.g., 102,103 and 104 in FIG. 1B). Primers of first set (1212) anneal to V regions(1226) of recombined nucleic acids (1200) and primers of first set(1212) are extended (1202) through D region (1224) into at least Jregion (1222) and optionally to C region (1220) to form first extensionproducts (1216) that includes optional sequence tag (1228) and firstprimer binding site (1230). After removing nonextended primers of firstset (1212), primers of second set (1240) are added to the reactionmixture tinder annealing conditions so that they anneal to theirrespective target J regions (1222), after which they are extended (1204)to form second extension products (1232), each of which comprisessequence tag (1236) (optional) and second primer binding site (1234).Second extension products (1232) may comprise a single sequence taglocated, for example, adjacent to V regions (1226), as shown by sequencetag (1228), or adjacent to J regions (1222), as shown by sequence tag(1236), or second extension products (1232) may comprise two sequencetags located in both positions. In one embodiment, second extensionproducts (1232) comprise a single sequence tag (1228) adjacent to Vregions (1226). In another embodiment, second extension products (1232)comprise a single sequence tag (1236) adjacent to J regions (1224). Insome embodiments, sequence tags (1228) and/or (1236) are mosaic tagsdescribed below. After nonextended primers of second set (1240) areremoved, common forward and reverse primers are added which are specificfor first and second primer binding sites (1230) and (1234),respectively, and a PCR is carried out (1206). A sample of the resultingamplicon is sequenced (1208) to generate sequence reads for constructingclonotypes and clonotype profiles.

FIG. 1D illustrates a method of generating templates of a defined lengthand for attaching one or two sequence tags thereto. The embodiment ofFIG. 1D shows messenger RNA (mRNA) as the starting material, but themethod may be used with either DNA or RNA samples. To mRNA (1300)containing a VDJ region, one or more primers (1312) specific for Cregion (1308) (“C primers”) are annealed to mRNA (1300). Usually only asingle C primer is used. Alternatively, one or more primers (having asimilar structure) specific for J region may be used. C primer (1312)comprises target specific segment (1313), sequence tag segment (1314)and common primer binding site (1315). Also annealed to target mRNAs(1300) are polymerase blockers (1310), which may oligonucleotidesspecific for V regions (1302). In some embodiments, blockers (1310) maybe a natural oligonucleotide so long as a polymerase used to extendprimer (1312) does not have either strand displacement activity or 5′→3′exonuclease activity and so long as the oligonucleotide isnon-extendable, e.g., it has a 3′-deoxynucleotide. Usually, blockers(1310) are oligonucleotide analogs with enhanced binding activity andnuclease resistance, such as antisense compounds. In some embodiments,blockers (1310) may be locked nucleic acids (LNAs) or peptide nucleicacids (PNAs) or bridged nucleic acids (BNAs), which are disclosed in thefollowing references. Wengel et al, U.S. Pat. Nos. 6,794,499, 7,572,582;Vester et al, Biochemistry, 43(42): 13233-13241 (2004); and the like,and Kazuyuki et al, Chem. Comm., 3765-3767 (2007); Nielson et al, Chem.Soc. Rev. 26: 73-78 (1997); and the like. Sequences of blockers (1310)are selected so that the extension of primer(s) (1312) are halted at apredetermined location on V region (1302). In some embodiments, blockers(1310) are designed so that only enough of V region (1302) is copied inthe extension step so that the V region can be identified from thecopied sequence. In some embodiments, obtaining blockers (1310) specificfor each V region is unnecessary, as consensus sequences may be selectedthat permit some mismatches, so long as the progression of a polymeraseis stopped. The lengths of blockers (1310) may vary widely depending onthe kind of oligonucleotide or analog used. In some embodiments,blockers (1310) have lengths in the range of from 10 to 25 monomers. Insome embodiments, blockers (1310) may anneal to different locations ondifferent V region sequences.

Returning to FIG. 1D, primers (1312) are extended to blockers (1310)making a cDNA copy of a portion of VDJ region of target (1300) that hasa predetermined length. In some embodiments, the predetermined length(or equivalently the binding sites of blockers (1310)) are selected sothat a desired portion of the VDJ region may be covered by one or moresequence reads of the sequence technique used in the method. Afterextension is completed, RNA template (1300) is digested (1325) usingconventional techniques, e.g. digestion with an RNAse, such as RNAseHand/or RNAse A, to give single stranded cDNA (1326). To this cDNA isadded a 3′ mononucleotide tail, such as a polyC tail, using terminaldeoxynucleotide transferase (TdT) in a conventional protocol. To tailedcDNA (1331), adaptor (1336) having a complementary overhang to themononucleotide tail of cDNA (1331), after which it is extended toproduce double stranded DNA (1340), which may be amplified, e.g., by PCR(1337), and the resulting amplicon sequenced (1338).

Recombined nucleic acids that undergo hypermutation, such asIgH-encoding nucleic acids, may be amplified using sets of primers thatinclude primers that bind to different primer binding sites on the samerecombined nucleic acid; that is, such sets May include primers thatbind to one or more nonoverlapping primer binding sites on the samerecombined nucleic acid encoding a receptor chain. Such set may compriseone or both first set of primers and second set of primers. In someembodiments, recombined nucleic acids subject to hypermutation areamplified with a first set of primers and a second set of primerswherein at least one of the two sets comprises primers specific for aplurality of nonoverlapping primer binding sites, for example, one setmay contain for each different V segment a plurality of primers eachspecific for a different nonoverlapping Kibler binding on the differentV segments. An embodiment applicable to amplification of recombinednucleic acids undergoing hypermutation is illustrated in FIGS. 3A-3B,where nested sets of primers are employed to ensure amplification ofeach recombined nucleic acid in a sample under conditions, for example,of somatic hypermutation, clonal evolution, or the like. Recombinednucleic acids, e.g., encoding IgH molecules, are combined in a reactionmixture under annealing conditions with first nested set (302) ofprimers, comprising in this example groups (304), (306) and (308) ofprimers specific for different sites along V region (316) of recombinednucleic acids (300). In this embodiment, the first nested set comprisesa plurality of groups of primers, each specific for a different site orlocation of the V region, wherein the different members of a group arespecific for different variants of the region at the site. In someembodiments, the plurality of groups is in the range of 2-4; in otherembodiments, the plurality is 2 or 3. In some embodiments, each primerof first nested set (302) may have a unique sequence tag (314) and firstprimer binding site (312) in a 5′ non complementary tail. Primers offirst nested set (302) anneal to their target recombined nucleic acidsand are extended through D region (318) and at least a portion of Jregion (320) to form first amplicon (323) which comprises threecomponents (330), (332) and (334) corresponding to the three subsets orprimers (304), (306) and (308), respectively. Each member of firstamplicon (323) incorporates sequence tag (324) and printer binding site(326).

After nonextended primers are removed (322), second nested set ofprimers (340) is added the reaction mixture under annealing conditions.As illustrated in FIG. 3A, printers of second nested set (340) comprisesubsets (336) and (338) a primers which anneal at differentnonoverlapping positions on J region (320) of members of first amplicon(323). In some embodiments, the second nested set of primers may containonly a single group of primers. Primers of second nested set (340) areextended to form second extension product (360) which comprises subsets(350), (352) and (354) which, in turn, each comprise two further subsets(subsubsets) corresponding to primers (336) and (338). In someembodiments, second nested set of primers (340) contain printersspecific to only a single primer binding site and first nested set ofprimers (302) contain primers specific to at least two non-overlappingprimer binding sites. After removing nonextended primers (342), commonforward and reverse primers may be added to carry out PCR (356) and asample of the resulting amplicon may be sequenced (358). In variousembodiments, primers of both the first nested set and the second nestedset may include sequence tags (339); primers of the first nested set butnot the second nested set may include sequence tags; and primers of thesecond nested set but not the first nested set may include sequencetags. In some embodiments, primers of the first nested set are extendedfirst after which non-extended primers are removed or destroyed andprimers of the second nested set are annealed and extended (asillustrated in FIGS. 3A-3B). In other embodiments, the order of theannealing, extending and removing steps are reversed; that is, primersof the second nested set are extended first after which non-extendedprimers are removed or destroyed and primers of the first nested set areannealed and extended.

In some embodiments of the above method, more than one extension step,either (322) or (342), may be implemented, for example, in order toattach sequence tags to a greater fraction of target polynucleotides ina sample. In such embodiments, more than one distinct sequence tag maybe attached to a target polynucleotide and/or copies thereof. That is, aplurality of different sequence tags may be attached to a targetpolynucleotide and its progeny from an amplification reaction, such asPCR; thus, copies of an original target polynucleotide may be labeledwith more than one sequence tag. As explained more fully below, suchpluralities of sequence tags are still useful in tracking carry overcontamination and in permitting more sensitive determination of targetpolynucleotide sequences.

Some of the embodiments described above may be carried out with thefollowing steps. For example, a method of generating clonotype profilesfrom multiple, or a plurality of, T cell receptor chains may comprisethe steps of: (a) combining in a reaction mixture under primer extensionconditions a first set of primers with a sample of recombined nucleicacids from T-cells, wherein each primer of the first set has areceptor-specific portion with a length such that the receptor-specificportion anneals to a different recombined nucleic acid at apredetermined location or site one the target recombined nucleic acidand is extended to form a first extension product, and wherein eachprimer of the first set has a 5′-non-complementary end containing afirst primer binding site; (b)) removing front the reaction mixturenon-extended printers of the first set; (c) adding to the reactionmixture under primer extension conditions it second set of primers,wherein each primer of the second set has a receptor-aspect fie portionsuch that the receptor-specific portion anneals to the first extensionproduct at a predetermined location or site and has a5′-non-complementary end containing a second primer binding site,primers of the first set and/or primers of the second set comprising asequence tag disposed between the receptor-specific portion and thefirst or second primer binding site, respectively, and wherein eachprinter of the second set is extended to form a second extensionproduct, such that each second extension product comprises a firstprimer binding site, a second primer binding site, at least one sequencetag, and either (i) a portion of a Vβ segment and a portion of a Jβsegment of a T cell receptor chain, (ii) a portion of a Vδ segment and aportion of a J δ segment of a T cell receptor chain, or (iii) a portionof a Vγ segment and a portion of a Jγ segment or a T cell receptorchain; (d) performing a polymerase chain reaction in the reactionmixture to form an amplicon, the polymerase chain reaction using forwardprimers specific for the first primer binding site and reverse primersspecific for the second primer binding site; and (e) sequencing thenucleic acids of the amplicon to form a clonotype profile of multiple Tcell receptor chains. As used herein, “primer extension conditions” in areaction mixture includes conditions in which substantially all primerbinding sites are in a single stranded state. In some embodiments, suchconditions are obtained by melting double stranded target nucleic acidso that printer binding sites are in single stranded form so that primerscan anneal to them to form substrates for polymerase extension.

The predetermined locations or sites at which primers of the first andsecond sets bind may be determined by conventional methods known tothose of ordinary skill in the art of multiplex nucleic acidamplifications, such as multiplex PCRs, as exemplified in the referencescited below. For example, in the case of target polynucleotides beingrecombined nucleic acids encoding immune receptor molecules, Faham andWillis (cited above), Van Dongen et al, Leukemia, 17: 2257-2317 (2003),and like references provide guidance for selecting primer binding sitesfor multiplex amplification of such target polynucleotides. In someembodiments, selecting such predetermined locations or sites depends ofseveral factors including (i) their effect on amplification efficiency(it is desirable that frequencies of different copies in an ampliconfaithfully represent frequencies of target polynucleotides in tosample), (ii) their effect on the lengths of copies in an ampliconcorrespond to requirements of the DNA sequencing Chemistry beingemployed, (iii) whether the selected primers span a portion of therecombined nucleic acids with desired diversity, e.g. a VDJ region, andthe like. In relation to this aspect, in part the invention includes anappreciation and recognition that primer cross-reactivity with differenttarget polynucleotides does not effect results of methods of theinvention as compared to, for example, in methods based solely on analogreadouts of PCR amplifications, spectra typing, and the like), because aset of sequences is the readout rather than an analog signal.

In some embodiments, the step or sequencing includes the followingsteps: (i) providing a plurality of sequence reads each having an errorrate and each comprising a nucleotide sequence and a tag sequence, and(ii) aligning groups of sequence reads having like tag sequences, afterwhich base calls are made based on sequence reads within the groups todetermine the nucleotide sequence. Such group-level nucleotide sequencesmay then be coalesced into the same or different clonotypes as describedbelow. In some embodiments, in the PCR steps, the lengths of thereceptor-specific portions of the primers of the first and second setsare selected so that relative levels of different recombined nucleicacids in the amplicon are substantially the same as those of recombinednucleic acids in the sample. In implementing such selection of primersthe positions and lengths of the binding rites of the primers on theirrespective target polynucleotides may be varied. In some embodiments,sequence tags are selected from a set of sequence tags winch is muchlarger than the number of distinct target polynucleotides in a sample,so that substantially every distinct target polynucleotide in the sampleand copies thereof will have a different sequence tag (for example, inaccordance with the “labeling by sampling” methodology described inBrenner, U.S. Pat. No. 7,537,897). In some embodiments, the number ofsequence tags in such a set is at least 100 times the size of thepopulation of target polynucleotides in a sample. Further, in someembodiments where substantially every original target polynucleotide andcopies thereof are labeled with the same unique sequence tag, the stepof sequencing includes generating sequence reads of nucleic acids of theamplicon and aligning sequence reads having the same sequence tags todetermine sequence reads corresponding to the same clonotypes thesample. Further, in some embodiments, the step of aligning furtherincludes determining a nucleotide sequence of each clonotype bydetermining a majority nucleotide at each nucleotide position of thesequence reads having the same sequence tag. Further, in someembodiments, steps of removing the non-extended primers may be carriedout by digesting single stranded nucleic acids in the reaction mixtureusing a nuclease having 3′→5′ single strand exonuclease activity (whichmay be provided by, for example, E. coli exonuclease 1, which may beconveniently inactivated by heat). In further embodiments, the abovemethods may be used to generate clonotype profiles for diagnosing and/ormonitoring minimal residual disease of a cancer patient, such as amyeloma, lymphoma or leukemia patient. Such diagnosing and/or monitoringmay be implemented with the following additional step after the abovemethod steps: determining from the clonotype profile a presence, absenceand/or level of one or more patient-specific clonotypes correlated withthe cancer. Methods of this embodiment may further include steps ordetermining sequences of each of one or more sequence tags and comparingsuch sequences with sequences of sequence tags of previously determinedclonotype profiles to determine a presence, absence and/or level ofcontaminating sequences. In some embodiments, such step of comparingincludes comparing the sequences of one of more sequence tags tosequence tags of a clonotype database containing clonotypes from atleast one individual other than the patient.

In still another embodiment, a method of amplifying in one reaction aplurality of recombined nucleic acids encoding β, δ and γ T cellreceptor components may comprise the steps of: (a) combining in areaction mixture under primer extension conditions a first set ofprimers with a sample of recombined nucleic acids from T-cells, whereineach of the recombined nucleic acids comprises at first end at least aportion of a Jβ, Jδ or Jγ segment of a T cell receptor, and wherein eachprimer of the first set each has a receptor-specific portion with alength, which receptor-specific portion anneals to the first end of adifferent recombined nucleic acid and is extended to form a firstextension product, and wherein each printer of the first set has a5′-non-complementary end containing in a 3′→5′ ordering a sequence tagand a first primer binding site, the sequence tag being different forsubstantially every primer of the first set; (b) removing from thereaction mixture non-extended primers of the first set; (c) adding tothe reaction mixture under primer extension conditions a second set ofprimers, each primer of the second set having a receptor-specificportion with a length, which anneals to the first extension product andis extended to form a second extension product, wherein each secondextension product comprises at least a portion of a Vβ, Vδ or Vγ segmentof a T cell receptor, and wherein each primer of the second set has a5′-non-complementary end containing a second primer binding site; and(d) performing polymerase chain reaction in the reaction mixture to forman amplicon, the polymerase chain reaction using a forward primerspecific for the first primer binding site and a reverse primer specificfor the second primer binding site. The above method may further includea step of sequencing a sample of sequences of the amplicon. Typicallysuch sample as a “representative sample” in that it is large enough tothat different clonotypes are present in the sample in approximately thesame frequencies as in the original sample of biological material. Insome embodiments, the step of sequencing includes providing a pluralityof sequence reads each having an error rate and each comprising anucleotide sequence and a tag sequence, and aligning sequence readshaving like tag sequences to determine sequence reads corresponding tothe same clonotype. Such sequence reads may be processed in further stepof coalescing, as described more fully below, whenever multiple sequencetags are attached to original target polynucleotides or copies thereof.

In to another embodiment, a method of generating clonotype profiles frommultiple T cell receptor chains may comprise the steps of: (a) combiningin a reaction mixture under primer extension conditions a first set ofprinters with a sample of recombined nucleic acids from T-cells, whereineach primer of the first set has a receptor-specific portion with alength such that the receptor-specific portion anneals to a differentrecombined nucleic acid at a predetermined location and is extended toform a first extension product, and wherein each primer of the first sethas a 5′-non-complementary end containing a first printer binding site;(b) removing front the reaction mixture non-extended printers of thefirst set; (c) adding, to the reaction mixture a second set of primers,wherein each primer of the second set has a receptor-specific portionwith a length, the receptor-specific portion being specific for thefirst extension product at a predetermined location and having a5′-non-complementary end containing a second printer binding site,primers of the first set and/or primers of the second set comprising asequence tag disposed between the receptor-specific portion and thefirst or second primer binding site, respectively; (d) performing afirst polymerase chain reaction to form a first amplicon, the firstpolymerase chain reaction using forward primers specific for the firstprimer binding site and primers of the second set, wherein eachnucleotide sequence of the first amplicon comprises a first printerbinding site, a second primer binding site, at least one sequence tag,and either a portion of a Vβ segment and a portion of a Jβ segment of aT cell receptor chain, a portion of a Vδ segment and a portion of a Jδsegment of a T cell receptor chain, or a portion of a Vγ segment and aportion of a Vγ segment of a T cell receptor chain, and wherein thelengths of the receptor-specific, portions of the primers of the firstand second sets are selected so that relative levels of differentrecombined nucleic acids in the amplicon are substantially the same asthose of different recombined nucleic acids in the sample; (e) addingreverse primers specific for the second primer binding site; (f)performing a second polymerase chain reaction in the reaction mixture toform a second amplicon, the polymerase chain reaction using forwardprimers specific for the first primer binding site and reverse primersspecific for the second primer binding site; (g) sequencing the nucleicacids of the second amplicon to form a clonotype profile of multiplecell receptor chains. In some embodiments, the step of sequencingincludes providing a plurality of sequence reads each having an errorrate and each comprising a nucleotide sequence and a to sequence, andaligning sequence reads having like tag sequences to determine sequencereads corresponding to the same clonotype. In further embodiments wheretarget polynucleotides and/or copies thereof are labeled with more thanone sequence tag, after aligning like sequence tags, sequence reads maybe processed in a further step of coalescing, as described more fullybelow.

In another example, a method of generating clonotype profiles frommultiple B cell receptor chains may be carried out by the steps of: (a)combining in a reaction mixture under printer extension conditions afirst nested set of primers with a sample of recombined nucleic acidsfrom B-cells, the first nested set comprising one or more groups ofprimers, wherein each primer of each group has a receptor-specificportion with a length such that the receptor-specific portion of eachprimer :from a different group anneals to a different recombined nucleicacid at a predetermined site that does not overlap a predetermined siteof any other primer of the first nested set, and wherein each primer ofeach group has a 5′-non-complementary end containing a first primerbinding site; (b) extending primers of the first nested set to loon afirst extension product; (c) removing from the reaction mixturenon-extended primers of the first nested set; (d) adding to the reactionmixture under primer extension conditions a second nested set ofprimers, the second nested set comprising one or more groups of primers,wherein each primer of each group has a receptor specific portion with alength such that the receptor-specific portion of each primer from adifferent group anneals to the first extension product at apredetermined site that does not overlap a predetermined site of anyother primer of the second nested set, and wherein each primer of eachgroup has a 5′-non-complementary end containing a second primer bindingsite, and wherein primers of the first nested set and/or primers of thesecond nested set comprise a sequence tag disposed between itsreceptor-specific portion and its first or second primer binding site,respectively: (e) extending primers of the second nested set to form asecond extension product, such that each second extension productcomprising a first primer binding site, a second primer binding site, atleast one sequence tag, and either (i) a portion of a V segment and aportion of a J segment of a B cell receptor heavy chain, or (ii) aportion of a V segment and a portion of a J segment of a B cell receptorkappa light chain; (f) performing a polymerase chain reaction in thereaction mixture to form an amplicon, the polymerase chain reactionusing forward primers specific for the first primer binding site andreverse printers specific for the second primer binding site and (g)sequencing the nucleic acids of the amplicon to form a clonotype profileof multiple B cell receptor chains.

In some embodiments, more than one cycles of annealing and extendingprimers (after melting the extension product) may be implemented insteps (b) and/or (e), in which case copies of the original recombinednucleic acids in the sample may be labeled with one or more sequencetags. In these embodiments, sequencing step (g) may include furthersteps of aligning and coalescing as described below for determiningclonotypes and clonotype profiles. In some embodiments, for example,where only single extensions are made in steps (b) and (e), the step ofsequencing includes providing a plurality of sequence reads each havingan error rate and each comprising a nucleotide sequence and a tagsequence, and aligning sequence reads having, like tag sequences todetermine sequence reads corresponding to the same clonotype. As abovein some embodiments, in the PCR the positions and the lengths of thereceptor-specific portions of the primers of the first and second setsare selected so that relative levels of different recombined nucleicacids in the amplicon are substantially the same as those of differentrecombined nucleic acids in the sample.

In some of the embodiments, sequence tags are attached to a targetpolynucleotide or a copy thereof in a step of primer extension, whereinsubstantially every different target polynucleotide and copy thereof islabeled with the same sequence tag. In other embodiments, targetpolynucleotides of a sample or copies thereof may be labeled with morethan one different sequence tags. As explained further below, in someembodiments, multiple extensions or multiple cycles of a PCR may becarried out in the presence of sequence tag-containing primers (either afirst set of primers or a second set of primers), which may result indifferent sequence tags being attached to the same target polynucleotideand/or its copies.

In one aspect, the invention is directed to a method for obtaining andanalyzing sequence data from at repertoire of immune molecules, such asT cell receptors (TCRs) or B cell receptors (BCRs) or defined fragmentsthereof, to rapidly and efficiently determine a clonotype profile.Sequence data typically comprises a large collection of sequence reads,i.e. sequences of base calls and associated quality scores, from a DNAsequencer used to analyze the immune molecules. A key challenge inconstricting clonotype profiles is to rapidly and accurately distinguishsequence reads that contain genuine differences from those that containerrors from non-biological sources, such as the extraction steps,sequencing chemistry, amplification chemistry, or the like. An aspect ofthe invention includes attaching a unique sequence tag to each targetpolynucleotide, for example, recombined nucleic acid, in a sample toassist in determining whether sequence reads of such conjugates arederived from the same original target polynucleotide. In accordance withone aspect of the invention, sequence tags are attached to thesomatically recombined nucleic acid molecules to form tag-moleculeconjugates wherein each recombined nucleic acid of such a conjugate hasa unique sequence tag. Usually such attachment is made after nucleicacid molecules are extracted from a sample containing T cells and/or Bcells and/or cell-free DNA. Preferably, such unique sequence tags differas greatly as possible from one another as determined by conventionaldistance measures for sequences, such as a Hamming distance, or thelike. By maximizing the distance between sequence tags in tag-moleculeconjugates, even with at high rate of sequencing and amplificationerrors, a sequence tag of a conjugate remains far closer to itsancestral tag sequence than to that of any other tag sequence of adifferent conjugate. For example, if 16-mer sequence tags are employedand each such tag on a set of clonotypes has a Hamming distance of atleast fifty percent, or eight nucleotides, from every other sequence tagon the clonotypes, then at least eight sequencing or amplificationerrors would, be necessary to transform one such tag into another for amiss-read of a sequence tag (and the incorrect grouping of a sequenceread of a clonotype with the wrong sequence tag). In one embodiment,sequence tags are selected so that after attachment to recombinednucleic acids molecules to form tag-molecule conjugates, the Hammingdistance between tags of the tag-molecule conjugates is a number atleast twenty-five percent of the total length of such sequence tags thatis, each sequence lag differs in sequence from every other such tag inat least 25 percent of its nucleotides in another embodiment, theHamming, distance between such sequence tags is a number at least 50percent of the total length of such sequence tags.

In one aspect, the invention is implemented by the following steps: (a)obtaining a sample from an individual comprising T-cells and/or B-cellsand/or cell-free DNA: (b) attaching sequence tags to molecules ofrecombined nucleic acids of T-cell receptor genes or immunoglobulingenes in the sample to form tag-molecule conjugates, whereinsubstantially every molecule of the tag-molecule conjugates has a uniquesequence tan: (e) amplifying the tag-molecule conjugates; (d) sequencingthe tag-molecule conjugates; and (e) aligning sequence reads of likesequence tags to determine sequence reads corresponding to the samerecombined nucleic-acid in the sample. Samples containing B-cells orT-cells are obtained using conventional techniques. In the step ofattaching sequence tags, preferably sequence tags are not only uniquebut also are sufficiently different from one another that the likelihoodof even a large number of sequencing or amplification errorstransforming one sequence tag into another would be close to zero. Afterattaching sequence tags, amplification of the tag-molecule conjugate isnecessary for most sequencing technologies; however, wheneversingle-molecule sequencing technologies are employed an amplificationstep is optional. Single molecule sequencing technologies include, butare not limited to, single molecule real-time (SMRT) sequencing,nanopore sequencing, or the like, e.g. U.S. Pat. Nos. 7,313,308,8,153,375; 7,907,800; 7,960,116; 8,137,569; Manrao et al, NatureBiotechnology, 4(8): 2685-2693 (2012); and the like.

In another aspect, the invention includes method for determining thenumber of lymphocytes in a sample by counting unique sequence tags. Evenwithout sequence tags, clonotypes of TCRβ or IgH genes, particularlythose including the V(D)J regions, provide for a lymphocyte and itsclones a unique marker. Whenever recombined nucleic acids are obtainedfrom genomic DNA, then a count of lymphocytes in a sample may beestimated by the number of unique clonotypes that are counted aftersequencing. This approach breaks down whenever there are significantclonal populations of identical lymphocytes associated with the sameclonotype for when recombined nucleic acids are obtained front mRNA of asample, whose quantity of individual sequences may reflect, or dependon, expression rate as well as cell number). The use of sequence tagsovercomes this short coming and is especially useful for providingcounts of lymphocytes in patients suffering from many lymphoiddisorders, such as lymphomas or leukemias. In accordance with one aspectof the invention, sequence tags may be used to obtain an absolute countof lymphocytes in a sample regardless of whether there is a largedominant clone present, such as with leukemia. Such a method may beimplemented with the steps: (a) obtaining a sample from an individualcomprising lymphocytes; (b) attaching sequence tags to molecules ofrecombined nucleic acids of T-cell receptor genes or of immunoglobulingenes of the lymphocytes to form tag-molecule conjugates, whereinsubstantially every molecule of the tag-molecule conjugates has a uniquesequence tag; (c) amplifying the tag-molecule conjugates: (d) sequencingthe tag-molecule conjugates; and (e) counting the number of distinctsequence tags to determine the number of lymphocytes in the sample. Insome embodiments, the molecules of recombined nucleic acids are fromgenomic DNA.

In one embodiment of the invention, sequence tags are attached torecombined nucleic acid molecules of a sample by labeling by sampling,as disclosed by Brenner et al, U.S. Pat. No. 5,846,719; Brenner et al,U.S. Pat. No. 7,537,897; Macevicz, International patent publication WO2005/111242; and the like, which are incorporated herein by reference.In labeling by sampling, polynucleotides of a population to be labeled(or uniquely tagged) are used to sample (by attachment, linking, or thelike) sequence tags of a much larger population. That is, if thepopulation of polynucleotides has K members (including, replicates ofthe same polynucleotide) and the population of sequence tags has Nmembers, then N>>K. In one embodiment, the size of a population ofsequence tags used with the invention is at least 10 times the size ofthe population of clonotypes in a sample; in another embodiment, thesize of a population of sequence tags used with the invention is atleast 100 times the size of the population of clonotypes in a sample;and in another embodiment, the size of a population of sequence tagsused with the invention is at least 1000 times the size of thepopulation of clonotypes in a sample. In other embodiments, a size ofsequence tag population is selected so that substantially everyclonotype in a sample will have a unique sequence tag whenever suchclonotypes are combined with such sequence tag population, e.g. in anattachment reaction, such as a ligation reaction, amplificationreaction, or the like. In some embodiments, substantially everyclonotype means at least 90 percent of such clonotypes will have aunique sequence tag; other embodiments, substantially every clonotypemeans at least 99 percent of such clonotypes will have a unique sequencetag. In other embodiments, substantially every clonotype means at least99.9 percent of such clonotypes will have a unique sequence tag. In manytissue samples or biopsies the number of T cells or B cells may be up toor about 1 million cells; thus, in some embodiments of the inventionemploying such samples, the number of unique sequence tags employed inlabeling by sampling is at least 10⁸ or in other embodiments at least10⁶.

In such embodiments, in which up to 1 million clonotypes are labeled bysampling, large sets of sequence tags may be efficiently produced bycombinatorial synthesis by reacting a mixture of all four nucleotideprecursors at each addition step of a synthesis reaction, e.g. asdisclosed in Church, U.S. Pat. No. 5,149,625, which is incorporated byreference. The result is a set of sequence tags hiving a structure of“N₁N₂ . . . N₈” where each N₁=A, C, G or T and k is the number ofnucleotides in the tags. The number of sequence tags in a set ofsequence tags made by such combinatorial synthesis is 4^(k). Thus, a setof such sequence tags with k at least 14, or k in the range of about 14to 18, is appropriate for attaching sequence tags to a 10′-memberpopulation of molecules by labeling by sampling. Sets of sequence tagswith the above structure include many sequences that may introducedifficulties or errors while implementing the methods of the invention.For example, the above combinatorially synthesized set of sequence tagsincludes many member tags with homopolymers segments that somesequencing approaches, such as sequencing-by-synthesis approaches, havedifficulty determining with accuracy above a certain length. Therefore,the invention includes combinatorially synthesized sequence tags havingstructures that are efficient for particular method steps, such assequencing. For example, several sequence tag structures efficient forsequencing-by-synthesis chemistries may be made by dividing the fournatural nucleotides into disjoint subsets which are used alternativelyin combinatorial synthesis, thereby preventing homopolymer segmentsabove a given length. For example, let z be either A or C and x beeither G or T to give a sequence tag structure of

[(z)_(t)(z)₂ . . . (z)_(i)][(x)_(t)(x)₂ . . . (x)_(j)]

where i and j, which may be the same or different, are selected to limitthe size of any homopolymer segment. In one embodiment, i and j are inthe range of from 1 to 6. In such embodiments, sequence tags may havelengths in the range of from 12 to 36 nucleotides; and in otherembodiments, such sequence tags may have lengths in the range of from 12to 24 nucleotides. In other embodiments other pairing of nucleotides mayused, for example, z is A or T and x is G or C; or z is A or G and x isor C. Alternatively, let be any combination of three of the four naturalnucleotides and let x′ be whatever nucleotide is not a z′ (for example,is A, C or G, and x′ is T). This gives a sequence tag structure asfollows:

[(z′)_(t)(z′)₂ . . . (z′)_(i) ]x′[(z′)_(t)(z′)₂ . . . (z′)_(i) ]x′

where i is selected as above and the occurrence of x′ serves as apunctuation to terminate any undesired homopolymers.

Further Sequence Tags

The invention uses methods of labeling nucleic acids, such as fragmentsof genomic DNA, with unique sequence tags, which may include “mosaictags,” prior to amplification and sequencing. Such sequence tags areuseful for identifying amplification and sequencing errors. Mosaic tagsminimize sequencing and amplification artifacts due to inappropriateannealing, priming, hairpin formation, or the like, that may occur withcompletely random sequence tags of the prior art. In one aspect, mosaictags are sequence tags that comprise alternating constant regions andvariable regions, wherein each constant region has a position in themosaic tag and comprises a predetermined sequence of nucleotides andeach variable region has a position in the mosaic tag and comprises apredetermined number of randomly selected nucleotides. By way ofillustration, a 22-mer mosaic tag (SEQ ID NO: 1) may have the followingform:

Region Position

There are nine constant and variable regions, with regions 1(nucleotides 1-3), 3 (nucleotide 9), 5 (nucleotides 12-14), 7(nucleotides 18-19) and 9 (nucleotides 21-22) being variable (doubleunderlined nucleotides) and regions 2 (nucleotides 4-8), 4 (nucleotides10-11), 6 (nucleotides 15-17), and 8 (nucleotide 20) being constant. Nrepresents a randomly selected nucleotide from the set of A, C, G or T;thus, the number of mosaic tags of this example is 4¹¹=4,194,304 tags. brepresents a predetermined nucleotide at the indicated position. In someembodiments, the sequence of b's, “***bbbbb*bb***bbb**b**”, is selectedto minimize the likelihood of having a perfect match in a genome of theorganism making up the sample.

In one aspect, for mosaic tags of a particular embodiment of the methodof the invention, all constant regions with the same position have thesame length and all variable regions with the same position have thesame length. This allows mosaic tags to be synthesized using partialcombinatorial synthesis with conventional chemistries and instruments.

In one aspect, mosaic tags comprise from 10 to 100 nucleotide; or 12 to80 nucleotides, or from 15 to 60 nucleotides. In some embodiments,mosaic tags comprise at least eight nucleotide positions with randomlyselected nucleotides; in other embodiments, whenever mosaic tags have alength of at least 15 nucleotides, they comprise at least 12 nucleotidepositions with randomly selected nucleotides. In another aspect, novariable region within a mosaic tag may have a length that is greaterthan seven nucleotides.

In another aspect, mosaic tags may be used in the following steps: (i)preparing DNA templates from nucleic acids in a sample; (ii) labeling bysampling the DNA templates to form a multiplicity tag-templateconjugates, wherein substantially every DNA template or a tag-templateconjugate has a unique mosaic tag comprising alternating constantregions and variable regions, each constant region having a position inthe mosaic tag and a length of from 1 to 10 nucleotides of apredetermined sequence and each variable region having a position in themosaic tag and a length of from 1 to 10 randomly selected nucleotides,such that constant regions having the satin positions have the samelengths and variable region having the same positions have the samelengths; (iii) amplifying the multiplicity of tag-template conjugates;(iv) generating a plurality of sequence reads for each of the amplifiedtag-template conjugates; and (v) determining a nucleotide sequence ofeach of the nucleic acids by determining a consensus nucleotide at eachnucleotide position of each plurality of sequence reads having identicalmosaic tags. In another aspect, mosaic tags may be used in the followingsteps: (a) preparing single stranded DNA templates from nucleic acids ina sample; (b) labeling by sampling the single stranded DNA templates toform tag-template conjugates, wherein substantially every singlestranded DNA template of a tag-template conjugate has a unique sequencetag (that is, a mosaic tag) having a length of at least 15 nucleotidesand having the following form:

[(N₁N₂ . . . N_(Kj))(b₁b₂ . . . b_(Lj))]M

wherein each N_(i) for i=1, 2, . . . K_(j), is a nucleotide randomlyselected from the group consisting of A, C, G and T; K_(j) is an integerin the range of from 1 to 10 for each j less than or equal to M (thatis, regions N₁N₂ . . . N_(Kj) are variable regions); each b_(i), fori=1, 2, . . . L_(j), is a nucleotide; L_(j) is an integer in the rangeof from 1 to 10 for each j less than or equal to M; such that everysequence tag (i) has the same Kj for every j and (ii) has the samesequences b₁b₂ . . . b_(i,j) for every j (that is, regions b₁b₂ . . .b_(i,j) are constant regions); and M is an integer greater than or equalto 2; (c) amplifying the tag-template conjugates; (d) generating asplurality of sequence reads for each or the amplified tag-templateconjugates; and (e) determining a nucleotide sequence of each of thenucleic acids by determining a Consensus nucleotide at each nucleotideposition of each plurality of sequence reads having identical sequencetags. In some embodiments, the plurality of sequence reads is at least10⁴; in other embodiments, the plurality of sequence reads is at least10⁵; in still other embodiments, the plurality or sequence reads is atleast 10⁶. In some embodiments, the total length of the above sequencetag is in the range of from 15 to 80 nucleotides.

Attaching Sequence Tags

A variety of different attachment reactions may be used to attach uniquetags to substantially every clonotype in a sample in addition to thoseillustrated above. Many techniques for capturing subsets of samplenucleic acids, for example, to reduce sample complexity in microrray orgenome sequencing technology, may be used with routine modification inthe present invention to attached sequence tags to recombined nucleicacids. Exemplary techniques for capturing diverse sets of target nucleicacids for subsequent manipulation, including attaching sequence tags,sequencing, and the like, include the following: Willis et al, U.S. Pat.No. 7,700,323: Jones et al, U.S. patent publication 2005/0142577;Gullberg et al, U.S. patent publication 2005/0037356: Porreca et al,Nature Methods, 4(11): 931-936 (2007); Turner et al, Nature Methods,6(5): 315-316 (2009); Church, U.S. Pat. No. 5,149,625: Macevicz, U.S.Pat. No. 8,137,936; and the like.

In one embodiment, such attachment is accomplished by combining a samplecontaining recombined nucleic acid molecules (which, in turn, compriseclonotype sequences) with a population or library of sequence tags sothat members of the two populations of molecules can randomly combineand become associated or linked, e.g. covalently. For example, suchrandom combining may occur in a bimolecular reaction wherein atag-containing primer anneals to a target nucleic acid and is extendedor wherein a tag-containing adaptor is ligated to the end of targetnucleic acid. In some embodiments, the method of attaching, tags maydepend in part on the DNA sequencing approach. For example, insequencing methods that produce relatively long accurate sequence reads,such as 454 sequencing, a cDNA library may be made from mRNA comprisingrecombined nucleic acids using conventional techniques, e.g. 5′-RACE,such as disclosed in Freeman et al, Genome Research, 19: 1817-1824(2009), after which sequence tags may be attached by ligatingsequence-tag-containing adapters to one or both ends. In otherembodiments, when sequencing methods, such as “Illumina” sequencing or“Ion Torrent” sequencing, are used that produce relatively short anderror-prone sequence reads, further steps may be required so thatamplicons for sequencing have lengths that are covered by sequence readsgenerated from the techniques. In such tag attachment reactions,clonotype sequences comprise linear single or double strandedpolynucleotides and sequence tags are carried by reagents such asamplification primers, such as PCP primers, ligation adapters,circularizable probes, plasmids, or the like. Several such reagentscapable of carrying sequence tag populations are disclosed in Macevicz,U.S. Pat. No. 8,137,936; Faham et al, U.S. Pat. No. 7,862,999; Landegrenet al U.S. Pat. No. 8,053,188; Carat and Deugau, Gene, 145 163-169(1994); Church, U.S. Pat. No. 5,149,625; and the like, which areincorporated herein by reference.)

FIGS. 2A and 2B illustrate an attachment reaction comprising a PCR inwhich is population of sequence tags (T₁, T₂, T₃ . . . T_(j), T_(j=1) .. . T_(k), T_(k=1) . . . T_(n−1), T_(R)) is incorporated into primers(2100). The population of sequence tags has a much greater size thanthat of recombined nucleic acid molecules (2102), The sequence rags areattached to the recombined nucleic acid molecules by annealing theprimers to the nucleic acid molecules and extending the primers with aDNA polymerase in the first cycle of a PCR. The figure depicts how therecombined nucleic acid molecules select, or sample, a small fraction ofthe total population of sequence tags by randomly annealing to theprimers by way of their common primer binding regions (2104), forexample, in V region (2108). Since the printers (an therefore sequencetags) combine with the recombined nucleic acid sequence moleculesrandomly, there is a small possibility that the same sequence tag may beattached to different nucleic acid molecules; however, if the populationof sequence tags is large as taught herein, then such possibility willbe negligibly small so that substantially every recombined nucleic acidmolecule will have a unique sequence tag attached. The other primer(2106) of the forward and reverse primer pair anneals to C region (2110)so that after multiple cycles of annealing, extending and melting,amplicon (2112) is formed, thereby attaching unique sequence tags to theV(D)J regions comprising the clonotypes of population (2102). That isamplicon (2112) comprises the tag-molecule conjugates from theattachment reaction.

FIGS. 2C and 2D illustrate a method for attaching a pair of sequencelags to each, or substantially each, recombined nucleic acid in asample. As in the method of FIGS. 2A and 2B, primers (2200) carryingsequence tags (T₁, T₂, T₃ . . . T_(j), T_(j=1) . . . T_(k), T_(k=1) . .. T_(n−1), T_(g)) are used as downstream printers and additionally,replacing common primer (2106), primers (2206) carrying sequence tags(Tm, Tm+1, Tm+2 . . . Tq+1, Tq+2, . . . Tr. Tr+1, Tr+2, . . . Ts, Ts+1,Ts+2, . . . ) are used as upstream primers. As with the downstream setof primers, the number of different sequence tags carried by upstreamprimers (2206) may be large compared to the number of recombined nucleicacid molecules (2202) so that substantially every recombined nucleicacid (2202) will have a unique tag after amplification, in someembodiments, each set of sequence tags in primers (2206) and (2200) neednot be as huge as the set of sequence tags in the embodiment of FIGS. 2Aand 2B. Since each recombined nucleic acid is uniquely labeled by a pairof sequence tags, sharing one sequence tag of the pair with a differencerecombined nucleic acid will not detract from the substantial uniquenessto a pair of sequence tans labelling a single recombined nucleic acid.Thus, in the embodiment of FIGS. 2C and 2D, sequence tags of each primerset (2200) and (2206) may be less diverse than the sequence tags ofprimer set (2100). For example, if random sequence tags are employed andprimers (2100) contain 16-mer sequence tags, then primers (2200) and(2206) may each contain 8-mer sequence tags to provide the same totalsequence tag diversity. Otherwise, the embodiment of FIGS. 2C and 2Doperates similarly to that of FIGS. 2A and 2B. Sequence tags ateattached to the recombined nucleic acid molecules by annealing theprimers to the nucleic acid molecules and extending the primers with aDNA polymerase in the first cycle of a PCR. As above, FIG. 2C depictshow the recombined nucleic acid molecules select, or sample, a smallfraction of the total population of pairs of sequence tags by randomlyannealing to the primers by way of their common primer binding regions(2204) and (2205), for example, in. V region (2218) and C region (2210),respectively. Since the primers (an therefore sequence tags) combinewith the recombined nucleic acid sequence molecules randomly, there is asmall possibility that the same pair of sequence tags may be attached todifferent nucleic acid molecules: however, if the population of sequencetags is large as taught herein, then such possibility will be negligiblysmall so that substantially every recombined nucleic acid molecule willhave a unique pair of sequence tags attached. After multiple cycles ofannealing, extending and melting, amplicon (2212) is formed, therebyattaching unique pairs of sequence tags to the V(D)J regions comprisingthe clonotypes of population (2202). That is amplicon (22112) comprisesthe tag-molecule conjugates from the attachment reaction.

In some embodiments, circularizable probes may be used to capture andattach sequence tags to desired recombined nucleic acids, for example,with routine modification of techniques disclosed by Porreca et at(cited above); Willis et al (cited above); or like references. Asillustrated in FIGS. 2E and 2F, circularizable probe (2302) is providedcomprising the following elements: upstream target binding segment(2304), downstream target binding segment (2306) that has5′-phosphorylated end (2305); sequence tag (2310); second common prunerbinding site (2314); optional cleavage site (2308); and first commonprimer binding site (2312), Circularizable probe (2302) is combined in areaction mixture under annealing conditions with a sample containingtarget polynucleotides (2300), which may be, for example, first orsecond strands of a cDNA prepared from mRNAs using conventionaltechniques. As shown, target polynucleotides comprise V, NDN, J and Cregions of recombined nucleic acids encoding IgHs or TCRβ chains. Insome embodiments, sequences of upstream and downstream target bindingsegments (2304) and (2306), respectively, are selected so that they spana portion of the VDJ region of the target polynucleotides.Circularizable probe (2302) and target polynucleotides (2300) formcomplex (2330) in the reaction mixture upon annealing of upstream anddownstream target binding segments (2304 and 2306). In the presence of aDNA polymerase and dNTPs, upstream target binding segment (2304) isextended (2340) to downstream target binding segment (2306) copying (andthereby capturing) a portion of the VDJ region of the targetpolynucleotide. In the presence of a ligase activity, the extendedupstream target binding segment is ligated to downstream target bindingsegment (2306), thereby forming a closed single stranded DNA circle(2342). The reaction mixture optionally may then be treated (2344) withan exonuclease to remove unreacted probe and target polynucleotides. Insome embodiments, single stranded circles (2342) are linearized bycleaving cleavage site (2308), which may be, for example, a rare-cuttingendonuclease recognition site, or inserting an RNA monomer in the probeand cleaving with RNase H or the like, after which VDJ-tag inserts ofthe linearized probes (2348) may be amplified by primers (2350) and(2352). Primers (2350) and (2352) may include non complementary regionsfor adding elements to permit later DNA sequencing (2354).Alternatively, single stranded circle may be used to generate nanoballtemplates for direct sequencing, e.g. Drmanac et al, Science, 327(5961);78-81 (2010); U.S. Pat. No. 8,445,196; and the like.

FIG. 23 illustrates another embodiment for attaching a sequence tag to arecombined nucleic acid encoding an immune receptor molecule. Guidancefor implementing this embodiment may be found in Faham and Zheng, U.S.Pat. No. 7,208,295, which is incorporated herein b reference. Recombinednucleic acids (2450) are combined in a reaction mixture under annealingconditions for probes (2454) and adapters (2456). Probes (2454) comprisereceptor-specific portion (2455) and adaptor-specific portion (2457).For example, probes (2454) may comprise a mixture of probes whereindifferent probes have receptor-specific portions specific for differentJ regions, or in other embodiments, specific for different V regions.Adapters (2456), which are 5′-phosphorylated, comprise probe-specificportion (2458) at its 5′ end, sequence tag (2460) and first primerbinding site (2462). The locations, sequences, and lengths ofreceptor-specific portion (2455) and adaptor-specific portion (2457) ofprobe (2454) and probe-specific portion (2458) are selected so that theyhybridize with one another to form structures (2452). After structure(2452) forms, single stranded portion (2461) is cleaved from recombinednucleic acid (2450) and the free 3′ end of recombined nucleic acid(2450) is ligated to the 5′ phosphorylated end of adaptor (2456) to formfirst extension product (2459), after which probe (2454) is removed(2474). Cleavage of (2461) may be effected by a single strandednuclease, as described in Faham and Zheng. In one embodiment, probe(2454) is synthesized with thymidines replaced with uracils, e.g. in aPCR with dUTPs in place or dTTps, and it is removed by treating withuracil-DNA glycosylase (UDG), e.g. as taught by Faham et al. U.S. Pat.No. 7,208,295, which is incorporated by reference. UDG treatment cleavesprobe (2454) at uracils to give fragments (2455). After free probe,adapters and flaps are removed (2476), forward primers (2466) andreverse primers (2468) are added to extension product (2464) and PCR(2470) is carried out, after which a sample of the resulting amplicon issequenced (2472).

In a similar embodiment to that of FIG. 2G, similar probes and adaptersmay be used to attached sequence tags at predetermined sites of a targetpolynucleotide, wherein a flap endonuclease, such as FEN-1, is used tocleave a single stranded portion corresponding to (2461). In thisembodiment besides using a different nuclease, the polarity of the probeand adaptor sequences are reversed; namely, a substrate for a flapendonuclease requires that the 3′ end of the adaptor corresponding to(2454) be annealed to target sequence (2450) and that the singlestranded portion corresponding to (2452) be a 5′ end of the targetsequence. After cleavage, and removal of the probe sequence, theremaining steps are substantially the same. Guidance for using flapendonucleases in detection assays may be found in the followingreferences: Lyamichev et al, Nature Biotechnology, 17: 292-296 (1999);Eis et al, Nature Biotechnology, 19: 673-676 (2001); and likereferences.

In some embodiments, recombined nucleic acids encode immune receptormolecule chains that typically form an immune repertoire which maycomprise a very law set of very similar polynucleotides (e.g. >1000, butmore than 10,000, and still more usually from 100,000 to 1,000,000, ormore) which may have a length of less than 500 nucleotides, or in otherembodiments, less than 400 nucleotides, or in still other embodiments,less than 300 nucleotides. In one aspect of the invention, the inventorsrecognized and appreciated that these characteristics permitted the useof highly dissimilar sequence tags to efficiently compare sequence readsof highly similar clonotypes to determine whether they are derived fromthe same original sequence or not.

Samples

The term “sample” refers to a quantity of biological material, which insome embodiments is obtained from a patient, and which contains cellsand/or cell-free DNA; that is, the term is used interchangeably with theterm “specimen,” or “tissue sample,” The term “sample” also sometimeused in a statistical sense of obtaining a subset, or portion, of alarger set or quantity, respectively, of, for example, recombinednucleic acids; in particular, the statistical usage of the term “sample”may also be understood to mean “representative sample,” in that such asample is understood to reflect, or approximate, the relativefrequencies of different nucleic acids in a tissue (for example). Oneskilled in the art is able to distinguish the proper usage from thecontext of the terms.

Clonotype profiles may be obtained from samples of immune cells orfluids, such as blood, containing cell-free nucleic acids encodingimmune receptor chains. For example, immune cells can include T-cellsand/or B-cells. T-cells (T lymphocytes) include, for example, cells thatexpress T cell receptors. T-cells include helper T cells (effector Tcells or Th cells), cytotoxic T cells (CTLs), memory T cells, andregulatory T cells. In one aspect a sample of T cells includes at least1,000T cells; but more typically, a sample includes at least 10,000 Tcells, and more typically, at least 100,000 T cells. In another aspect,a sample includes a number of T cells in the range of from 1000 to1,000,000 cells. A sample of immune cells may also comprise B cells,include, for example, plasma B cells, memory B cells B1 cells, B2 cells,marginal-zone B cells, and follicular B cells. B-cells can expressimmunoglobulins (antibodies, B receptor). As above, in one aspect asample of B cells includes at least 1,000 B cells; but more typically, asample includes at least 10,000 B cells, and more typically, at least100,000 B cells. In another aspect, a sample includes a number of Bcells in the range of from 1000 to 1,000,000 B cells.

Samples used in the methods of the invention can come from a variety oftissues, including, for example, tumor tissue, blood and blood plasma,lymph fluid, cerebrospinal fluid surrounding the brain and the spinalcord, synovial fluid surrounding bone joints, and the like. In oneembodiment, the sample is a blood sample. The blood sample can be about0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8. 0.9, 1.0, 1.5, 2.0, 2.5, 3.0,3.5, 4.0, 4.5, or 5.0 mL. The sample can be a tumor biopsy. The biopsycan be from, for example, from a tumor of the brain, liver, lung, heart,colon, kidney, or bone marrow. Any biopsy technique used by thoseskilled in the an can be used for isolating a sample from a subject. Forexample, a biopsy can be an open biopsy, in which general anesthesia isused. The biopsy can be a dosed biopsy, in which a smaller cut is madethan in an open biopsy. The biopsy can be a core or incisional biopsy,in which part of the tissue is removed. The biopsy can be an excisionalbiopsy, in which attempts to remove an entire lesion are made. Thebiopsy can be a fine needle aspiration biopsy, in which a sample oftissue or fluid is removed with a needle.

In some embodiments, clonotype profiles for methods of the invention aregenerated from a tumor or peripheral blood in the case of diagnosticsamples or from peripheral blood in the ease of samples for monitoringresidual disease. One or more clonotypes correlated with a disease, suchas a lymphoid or myeloid proliferative disorder, are determined from adiagnostic sample. Usually the one or more clonotypes correlated with alymphoid or myeloid proliferative disorder are those present in aclonotype profile with the highest frequencies. In some cases, theremaybe a single correlated clonotype and in other cases them may bemultiple clonotypes correlated with a lymphoid or myeloid proliferativedisorder. Tumor samples may be taken from any tissue affected by such adisorder, which includes lymph nodes or other tissues outside of thelymphatic system. As mentioned above, clonotype profiles for monitoringresidual disease may be generated from a sample of nucleic acidsextracted from peripheral blood. The nucleic acids of the sample mayfrom B-cells from a cell-containing fraction of the peripheral blood orfrom a cell free fraction of the peripheral blood, such as plasma orseruma. In one embodiment, a peripheral blood sample includes at least1,000 B cells; but more typically, such a sample includes at least10,000 B and more typically, at least 100,000 B cells. In anotheraspect, a sample, includes a number of B cells in the range of from 1000to 1,000,000 B cells. In some embodiments, the number of cells in asample sets a limit on the sensitivity of a measurement. That is,greater sensitivity of detecting a residual disease is achieved by usinga larger sample of peripheral blood. For example, in a sample containing1,000 B cells, the lowest frequency of clonotype detectable is 1/1000 or0.001, regardless of how many sequencing reads are obtained when the DNAof such cells is analyzed by sequencing. The nucleic acids of the samplemay from T-cells from a cell-containing fraction of the peripheral bloodor from a cell free fraction or the peripheral blood, such as plasma orserum. In one embodiment, a peripheral blood sample includes at least1,000 T cells; but more typically, such a sample includes at least10,000 T cells,, and more typically, at least 100,0001 cells. In anotheraspect, a sample includes a number of T cells in the range of from 1000to 1,000,000 T cells. In some embodiments, the number of cells in asample sets a limit on the sensitivity of a measurement. That is,greater sensitivity of detecting a residual disease is achieved by usinga larger sample of peripheral blood. For example, In a sample containing1,000 T cells, the lowest frequency of clonotype detectable is 1/1000 or0.001, regardless of how many sequencing reads are obtained when the DNAof such cells is analyzed by sequencing.

A sample for use with the invention can include DNA (e.g., genomic DNA)or RNA (e.g., messenger RNA). The nucleic acid can be cell-free DNA orRNA, e.g. extracted from the circulatory system, Vlassov et al, Curr.Mol. Med., 10: 142-165 (2010); Swamp et al, FEBS Lett., 581: 795-799(2007). In the methods of the provided invention, the amount of RNA orDNA from a subject that can be analyzed includes, for example, as low asa single cell in some applications (e.g., a calibration test with othercell selection criteria, e.g., morphological criteria) and as many as 10million of cells or more, which translates into a quantity of DNA in therange of from 6 pg-60 ug, and a quantity of RNA in the range of from 1pg-10 ug. In some embodiments, a nucleic acid sample is a DNA sample offrom 6 pg to 60 ug. In other embodiments, a nucleic acid sample is a DNAsample from 100 uL to 10 mL of peripheral blood: in other embodiments, anucleic acid sample is a DNA sample from a cell free fraction of from100 uL to 10 mL of peripheral blood.

In some embodiments, a sample of lymphocytes or cell free nucleic acidis sufficiently large so that substantially every B cell or T cell witha distinct clonotype is represented therein, thereby forming a“repertoire” of clonotypes. In one embodiment, to achieve substantialrepresentation of every distinct clonotype, a sample is taken thatcontains with a probability of ninety-nine percent every clonotype of apopulation present at a frequency of 0.001 percent or greater. Inanother embodiment, a sample is taken that contains with a probabilityof ninety-nine percent every clonotype of a population present at afrequency of 0.001 percent or greater. And in another embodiment, asample is taken that contains with as probability of ninety-nine percentevery clonotype of as population present at a frequency of 0.00001percent or greater. In one embodiment, a sample of B cells or T cellsincludes at least one half million cells, and in another embodiment suchsample includes at least one million cells.

Nucleic acid samples may be obtained front peripheral blood usingconventional techniques, e.g., Innis et al. editors, PCR Protocols(Academic Press, 1990); or the like. For example, white blood cells maybe separated from blood samples using convention techniques, e.g.RosetteSep kit (Stem Cell Technologies, Vancouver, Canada). Bloodsamples may range in volume from 100 μL to 10 mL; in one aspect, bloodsample volumes are in the range of front 100 μL to 2 mL. DNA and/or RNAmay then be extracted from such blood sample using conventionaltechniques for use in methods of the invention, e.g. DNeasy Blood &Tissue Kit (Qiagen, Valencia, Calif.). Optionally, subsets of whiteblood cells, e.g. lymphocytes, may be further isolated usingconventional techniques, e.g. fluorescently activated cell sorting(FACS)(Becton Dickinson, San Jose. Calif.), magnetically activated cellsorting (MACS)(Miltenyi Biotec, Auburn, Calif.), or the like. Forexample, memory B cells may be isolated by way of surface markers CD19and CD27.

Cell-free DNA may also be extracted from peripheral blood samples usingconventional techniques, e.g. Lo et al. U.S. Pat. No. 6,258,540; Huanget al, Methods Mol. Biol., 444: 203-208 (2008); and the like, which areincorporated herein by reference. By way of nonlimiting example,peripheral blood may be collected in EDTA tubes, after which it may befractionated into plasma, white blood cell, and red blood cellcomponents by centrifugation. DNA from the cell free plasma fraction(e.g. from 0.5 to 2.0 mL) may be extracted using a QIAamp DNA Blood MiniKit (Qiagen, Valencia, Calif.), or like kit, in accordance with themanufacturer's protocol.

In one aspect, a sample of lymphocytes for generating a clonotypeprofile is sufficiently large that substantially every T cell B cellwith a distinct clonotype is represented therein. In one embodiment, asample is taken that contains with a probability of ninety-nine percentevery clonotype of a population present at a frequency of 0.001 percentor greater. In another embodiment, a sample is taken that contains witha probability of ninety-nine percent every clonotype or a populationpresent at a frequency of 0.0001 percent or greater. In anotherembodiment, a sample is taken that contains with a probability ofninety-nine percent every clonotype a population present at a frequencyof 0.00001 percent or greater. In other embodiments, a sample is takenthat contains with a probability of ninety-five percent every clonotypeof a population present at a frequency of 0.001 percent or greater. Inanother embodiment, a sample is taken that contains with a probabilityof ninety-live percent every clonotype of a population present at afrequency of 0.0001 percent or greater. In another embodiment, a sampleis taken that contains with a probability of ninety-five percent everyclonotype d population present at a frequency of 0.0000.1 percent orgreater. In still another embodiment, a sample of B cells or cellsincludes at least a half million cells, and in another embodiment suchsample includes at least one million cells.

Whenever of source of Material from which a sample is taken is scarce,such as, clinical study sampler, or the like, DNA from the material maybe amplified by a non-biasing technique, such as whole genomeamplification (WGA), multiple displacement amplification (MDA); or liketechnique, e.g. Hawkins et al, Curr. Opin. Biotech., 13: 65-67 (2002);Dean et al, Genome Research, 11: 1095-1099 (2001); Wang et al, NucleicAcids Research, 32: e76 (2004); Hosono et al, Genome Research, 13:954-964 (2003); and the like.

Blood samples are of particular interested art may be obtained usingconventional techniques, e.g. Innis et al, editors, PRC Protocols(Academic Press, 1990); or the like. For example, white blood cells maybe separated from blood samples using convention techniques, e.g.RoseneSep kit (Stem Cell Technologies, Vancouver, Canada). Blood samplesmay range in volume from 100 μL to 10 mL; in one aspect, blood samplevolumes are in the range of from 100 μL to 2 mL. DNA and/or RNA may thenbe extracted from such blood sample using conventional techniques foruse in methods of the invention, e.g. DNeasy Blood & Tissue Kit (Qiagen,Valencia, Calif.). Optionally, subsets of white blood cells, e.g.lymphocytes, may be further isolated using conventional techniques, e.g.fluorescently activated cell sorting (FACS)(Becton Dickinson, San Jose,Calif.), magnetically activated cell sorting (MACS)(Miltenyl Biotec,Auburn, Calif.), or the like.

Since the identifying recombinations are present in the DNA of eachindividual's adaptive immunity cells as well as their associated RNAtranscripts, either RNA or DNA can be sequenced in the methods of theprovided invention. A recombined sequence from a T-cell or B-cellencoding a chain of a T cell receptor or immunoglobulin molecule, or aportion thereof, is referred to as a clonotype. The DNA or RNA cancorrespond to sequences from T-cell receptor (TCR) genes orimmunoglobulin (Ig) genes that encode antibodies. For example, the DNAand RNA can correspond to sequences encoding α, β, γ, or δ chains of aTCR. In a majority of T-calls, the TCR is a hetero dimer consisting ofan α-chain and β-chain. The TCRα chain is generated by VJ recombination,and the β chain receptor is generated by V(D)J recombination. For theTCRβ chain, in humans there are 48 V segments, 2D segments, and 13 Jsegments. Several bases may be deleted and others added (called N and Pnucleotides) at each of the two junctions. In a minority of T-cells, theTCRs consist of γ and δ delta chains. The TCR γ chain is generated by VJrecombination, and the TCR δ chain is generated by V(D)J recombination(Kenneth Murphy, Paul Travers, and Mark Walport, Janeway's Immunology7th edition, Garland Science, 2007, which is herein incorporated byreference in its entirety).

The DNA and RNA analyzed in the methods of the invention can correspondto sequences encoding heavy chain immunoglobulins (IgH) with constantregions (α, δ, ε, γ, or μ) or light chain immunoglobulins (IgK or IgL)with constant regions λ or κ. Each antibody has two identical lightchains and two identical heavy chains. Each chain is composed of aconstant (C) and a variable region. For the heavy chain, the variableregion is composed of a variable (V), diversity (D), and joining (J)segments. Several distinct sequences coding for each type of thesesegments are present in the genome. A specific VDJ recombination eventoccurs during the development of a B-cell, marking that cell to generatea specific heavy chain. Diversity in the light chain is generated in asimilar fashion except that there is no D region so there is only VJrecombination, mutation often occurs close to the site of therecombination, causing the addition or deletion of several nucleotides,further increasing the diversity of heavy and light chains generated byB-cells. The possible diversity of the antibodies generated by a B-cellis then the product of the different heavy and light chains. Thevariable regions of the heavy and light chains contribute to form theantigen recognition (or binding) region or site. Added to this diversityis a process of somatic hypermutation which can occur after a specificresponse is mounted against some epitope.

As mentioned above, in accordance with the invention, primers may beselected to generate amplicons containing portions of recombined nucleicacids from lymphocytes or from cell-free nucleic acids from a tissue,such as blood. Such portions may be referred to herein as “somaticallyrearranged regions.” Somatically rearranged regions may comprise nucleicacids from developing or from fully developed lymphocytes, wheredeveloping lymphocytes are cells in which rearrangement of immune geneshas not been completed to form molecules having (for example) full V(D)Jregions. Exemplary incomplete somatically rearranged regions includeincomplete IgH molecules (such as, molecules containing only D-Jregions), incomplete TCRδ molecules as, molecules containing only D-Jregions), and inactive IgK (for example, comprising Kde-V regions).

Amplification of Nucleic Acid Populations

In some embodiments, primer sequences of the first and second sets ofprimers may be selected in accordance with conventional multiplexpolymerase chain reactions (PCRs).. For example, guidance for selectingprimers and for carrying out multiplex PCRs of nucleic acids encodingvarious immune receptor chains is found in the following: references,which are in by reference: Faham and Willis, U.S. Pat. Nos. 8,236,503and 8,628927; Morley U.S. Pat. No. 5,296,351; Gorski, U.S. Pat. No.5,837,447; Dan, U.S. Pat. No. 6,087096; Van Dongen et al, U.S. patentpublication 2006/0234234; European patent publication EP 1544308B1; VanDoneen et al. Leukemia, 17: 2257-2317 (2003); and the like, Guidance formultiplex PCRs may be found in Henegarhu et al, BioTechniques, 23:504-511 (1997), and like references. In some embodiments, primers areselected so that frequencies of amplified sequences in a final productare substantially, the same as frequencies, of the sequences in thestarting reaction mixture. Such primer selection may include selectionof primer lengths, primer binding sites and primer concentrations. Asnoted above, depending on the methods selected to generate sequencereads and attached sequence tags, the level of multiplexing may varywidely.

In some embodiments, to step of amplifying target nucleic acids includeslinear amplification of target nucleic acids, such as, for example, byrepealed cycles of annealing one set of primers (for example, to firstset of “upstream” or “forward” primers), extending the primers, meltingthe extended strand from the template, so that the quantity of extendedstrands is amplified as a linear function of the number of cycles. Inother words, a step or amplifying include copying a targetpolynucleotide (that is, at least one strand of to targetpolynucleotide) by repeated extensions of one set of primers. In someembodiments, such single or repeated extensions in one direction may befollowed by steps of removing unextended primers and a single orrepeated extensions of another set of primers in the other direction(for example, as second set of “downstream” or “reverse” primers).

The number of primer in a first set of primers and a second set ofprimers may vary widely depending on the number and type of immunereceptor-chain nucleic acids are amplified in an assay. In someembodiments, consensus primers for various chains may be used. In otherembodiments, specific primers may he designed for each different targetpolynucleotide amplified. Usually, both first set and second set ofprimers each comprise a plurality of primers. In some embodiments, theplurality of primers in the first set or the second set of primers is atleast 50 primers; in other embodiments, the plurality of primers in thefirst set or the second set of primers Is at least 100 primers; in otherembodiments, the plurality of primers in the first set or the second setof primers is at least 150 primers; in other embodiments, the pluralityof primers in the first set or the second set of primers is at least 200primers; in other embodiments, the plurality of primers in the first setor the second set of primers is at least 250 primers. The number ofprimers in the first set may be the same or different than the number ofprimers are the second set.

In some embodiments, primers of the first set and the second set areselected so that the length of clonotypes are at least 30 nucleotides;in other embodiments, primers of the first set and the second set areselected so that the length of clonotypes are in the range of from 30 to500 nucleotides; in other embodiments, primers of the first set and thesecond set are selected so that the length of clonotypes are in therange of front 30 to 400 nucleotides. In other embodiments, primers ofthe first set and the second set are selected so that the length ofclonotypes are in the range of from 30 to 300 nucleotides; in otherembodiments, primers of the first set and the second set are selected sothat the length of clonotypes are in the range of from 30 to 200nucleotides.

Exemplary PCR amplification protocols may be found in van Dongen et al,Leukemia, 17: 2257-2317 (2003) or van Dongen et al, U.S. patentpublication 2006/0234234, which is incorporated by reference. Briefly,an exemplary protocol is as follows: Reaction buffer: ABI Buffer II orABI Gold Buffer (Life Technologies, San Diego, Calif.); 50 μL finalreaction volume; 100 ng sample DNA; 10 pmol of each primer (subject toadjustments to balance amplification as described below); dNIPs at 200μM final concentration; MgCl₂ at 1.5 mM final concentration (subject tooptimization departing on target sequences and polymerase); Taqpolymerase (1-2 U/tube); cycling conditious: preactivation 7 min at 95°C.; annealing at 60° C.; cycling times: 30s denaturation; 30s annealing;30s extension. Polymerases that can be used for amplification in themethods of the invention are commercially available and include, forexample, Taq polymerase, AccuPrime polymerase, or Pfu, The choice ofpolymerase to use can be based on whether fidelity or efficiency ispreferred.

Real time PCR, picogreen staining, nanofluidic electrophoresis (e.g.LabChip) or UV absorption measurements can be used in an initial step tojudge the functional amount of amplifiable material in a sample. In oneaspect, multiplex amplifications of the invention are carried out sothat relative amounts of sequences in a starting population aresubstantially the same as those in the amplified population, oramplicon. That is, multiplex amplifications are carried out with minimalamplification bias among member sequences of a sample population. In oneembodiment, such relative amounts are substantially the same if eachrelative amount in an amplicon is within five fold of its value in thestarting sample. In another embodiment, such relative amounts aresubstantially the same if each relative amount in an amplicon is withintwo fold of its value in the starting sample. As discussed more fullybelow, amplification bias in PCR may be detected and corrected usingconventional techniques so that a set of PCR primers may be selected fora predetermined repertoire that provide unbiased amplification of anysample.

In some embodiments, amplification bias may be avoided by carrying out atwo-stage amplification (for example, as described in Faham and Willis,cited above) wherein a small number of amplification cycles (forexample, 2-5, or 2-10, or 2-15 cycles) are implemented in a first, orprimary, stage using primers having tails non-complementary with thetarget sequences. The tails include primer binding sites that are addedto the ends of the sequences of the primary amplicon that such sites areused in a second stage amplification using only a single forward primerand a single reverse primer, thereby eliminating a primary cause ofamplification bias. Prior to initiation of the second stageamplification, non-extended printers of the first stage are removed fromthe reaction mixture., or are otherwise inactivated. In someembodiments, the primary PCR will have a small enough number of cycles(e.g. 2-10) to minimize the differential amplification by the differentprinters. The secondary amplification is then done with one pair ofprimers, which eliminates a source of differential amplification. Insome embodiments, a small percent or portion, e,g, one percent of thereaction volume, of the primary PCR taken directly to the secondary PCRreaction mixture. In some embodiments, a total of at least thirty-fivecycles allocated between a first stage amplification and a second stageamplification.

In some embodiments internal standards may be combined with andamplified in the same reaction as recombined nucleic acids of a sample.Internal standard are nucleic acids with known sequences and knownconcentrations. For example, they may be cloned copies of a naturalnucleic acid encoding portions of an immune receptor chain, or they maybe synthetic nucleic acids. In some embodiments, the lengths and basecompositions of the internal standards are selected to representative ofthe particular immune receptor chains being amplified. By monitoringchanges in the relative concentrations of the internal standards afteramplification, amplification bias may be detected, and conditions fornon-biased amplification may be determined. For example, primer lengths,positions, and concentrations may be varied to minimize bias in theamplification product. In some embodiments, a plurality of internalstandards are used in a reaction; in some embodiments, 2 to 50 differentinternal standards are used in a reaction; in other embodiments, from 2to 25 different internal standards are used in a reaction; and in someembodiments, front 2 to 10 different internal standards are used in areaction. In some embodiments, amplification bias is determined bymeasuring the relative, frequencies of the sequences of different targetnucleotides (for example, all or selected clonotypes internal standards)in an amplification product. In other embodiments, the presence, absenceor level of amplification bias may be determined by real-timequantitative PCR of selected nucleic acids, such as two or more theinternal standards. Internal, standards may also be used to quantify thenumbers of different: clonotypes in the original sample. Techniques forsuch molecular counting are well-known, e,g. Brenner et al, U.S. Pat.No. 7,537,897, which is incorporated herein by reference,

Generating Sentence Reads

Any high-throughput technique for sequencing nucleic acids can be usedhi the method of the invention. Preferably, such technique has acapability of generating in a cost-effective manner a volume of sequencedata from which at least 1000 clonotypes can be determined, andpreferably, from which at least 10,000 to 1,000,000 clonotypes can bedetermined. DNA sequencing techniques include classic dideoxy sequencingreactions (Sanger method) using labeled terminators or printers and gelseparation in slab or capillary, sequencing by synthesis usingreversibly terminated labeled nucleotides, pyrosequencing, 454sequencing, allele specific hybridization to a library of labeledoligonucleotide probes, sequencing by synthesis using allele specifichybridization to a library of labeled clones that is followed byligation, real time :monitoring or the incorporation of labelednucleotides during a polymerization step, polony sequencing, and solidsequencing. Sequencing of the separated molecules has more recently beendemonstrated by sequential or single extension reactions usingpolymerases or ligases as well as by single or sequential differentialhybridizations with libraries of probes. These reactions have beenperformed on many clonal sequences in parallel including demonstrationsin current commercial applications of over 100 million sequences inparallel. These sequencing approaches can thus be used to study therepertoire of T-cell receptor (TCR) and/or B-cell receptor (BCR). In oneaspect of the invention, high-throughput methods of sequencing areemployed that comprise a step of spatially isolating individualmolecules on a solid surface where they are sequenced in parallel. Suchsolid surfaces may include nonporous surfaces (such as in Solexasequencing, e.g. Bentley et al, Nature, 456; 53-59 (2008) or CompleteGenomics sequencing, e,g. Drmanac et al, Science, 327; 78-81 (2010)),arrays of wells, which may include bead- or particle-bound templates(such as with 454, e.g. Margulies et al, Nature, 437: 376-380 (2005) orIon Torrent sequencing; U.S. patent publication 2010/0137143 or2010/0304982), micromachined membranes (such as with SMRT sequencing,Eid et al. Science, 323: 133-138 (2009)), or bead arrays (as with solidsequencing or polony sequencing, e.g., Kim et al, Science, 316:1481-1414 (2007)). In another aspect, such methods comprise amplifyingthe isolated molecules either before or after they are spatiallyisolated on a solid surface. Prior amplification may compriseemulsion-based amplification, such as emulsion PCR, rolling circleamplification.

Of particular interest are approaches using sequencing by synthesis withreversible terminators, such as Solexa-based sequencing where individualtemplate molecules are spatially isolated on a solid surface., afterwhich they are amplified in parallel by bridge PCR to form separateclonal populations., or clusters, and then sequenced, such as describedin Bentley et al (cited above) and in manufacturer's instructions (e.g.TruSeq™ Sample Preparation Kit and Data Sheet, Illumina, San Diego,Calif., 2010); and further in the following references: U.S. Pat. Nos.6,190,512; 6,300,070; 7,115,400; and EP097208 B1; which are incorporatedby reference. In one embodiment, individual molecules disposed andamplified on a solid surface form clusters in a density of at least 10⁵clusters per cm²; or in a density of at least 5×10⁵ per cm²; or in adensity of at least 10⁶ clusters per cm². Solexa-based sequencing alsoprovides the capability of generating two sequence reads from the santetarget sequence (or template) in a cluster, one sequence read each fromopposite ends of a target sequence. In some embodiments, such pairs ofsequence reads may be combined and treated as a single sequence read insubsequent analysis, or such pairs may be treated separately but takinginto account that they originate from the same cluster. Sometimes thepair of sequence reads from the same template ate referred to as “matepairs,” and the process of sequencing from both ends of a temple isreferred to “bidirectional” sequencing. In some embodiments, a step ofsequencing by synthesis using reversibly terminated labeled nucleotidesincludes the generation of a single sequence read for each cluster orclonal population of templates and the generation of a plurality ofsequence roads (including but not limited to mate pairs) for eachcluster or clonal population of templates. In still further embodiments,when a plurality of sequence reads are generated for each cluster orclonal population of templates, such plurality of sequence reads may becombined to form a single effective sequence read that is used insubsequent analysis, such as a coalescing step.

In one aspect, a sequence-based clonotype profile of a sample front anindividual is obtained using the following step: (a) obtaining a nucleicacid sample from T-cells and/or B-cells of the individual; (b) spatiallyisolating individual molecules derived from such nucleic acid sample,the individual molecules comprising at least one template generated froma nucleic acid in the sample, which template comprises a somaticallyrearranged region or a portion thereof, each individual molecule beingcapable of producing at least one sequence read; (e) sequencing saidspatially isolated individual molecules; and (d) determining abundancesor different sequences of the nucleic acid molecules from the nucleicacid sample to generate the clonotype profile. In one embodiment, eachof the somatically rearranged regions comprise a V region and a Jregion. In another embodiment, the step of sequencing includesgenerating a plurality of sequence reads for each clonotype determined.In still other embodiments, the step of sequencing includes combininginformation or data from a plurality of sequence reads to form eachclonotype. In some embodiments, such step of combining may be carriedout by coalescing sequence reads as described in Faham and Willis, U.S.Pat. No. 8,628,927 (which is hereby incorporated by reference for thisteaching) or by using sequence tags as described in Faham et al, U.S.patent publication 2013/0236895A1 (which is hereby incorporated byreference for this teaching). In another embodiment, the step ofsequencing comprises bidirectionally sequencing each of the spatiallyisolated individual molecules to produce at least one forward sequenceread and at least one reverse sequence read,

Further to the latter embodiment, of least one of the forward sequencereads and at least one of the reverse sequence reads have an overlapregion such that bases of such overlap region are determined by areverse complementary relationship between such sequence reads. In stillanother embodiment, each of the somatically rearranged regions comprisea V region and a j region and the step of sequencing further includesdetermining a sequence of each of the individual nucleic acid moleculesfrom one or more of its forward sequence reads and at least one reversesequence read starting front a position in a region and extending it thedirection of its associated V region. In another embodiment, individualmolecules comprise nucleic acids selected from the group consisting ofcomplete IgH molecules, incomplete IgH molecules, complete IgK complete,IgK inactive molecules, TCRβ molecules, TCRγ molecules, complete TCRδmolecules, and incomplete TCRδ molecules. In another embodiment, thestep of sequencing comprises generating the sequence reads havingmonotonically decreasing quality scores. In another embodiment, theabove method comprises the following steps: (a) obtaining a nucleic acidsample from T-cells and/or B-cells of the individual; (b) spatiallyisolating individual molecules derived from such nucleic acid sample,the individual molecules comprising nested sets of templates eachgenerated from a nucleic acid in the sample and each containing asomatically rearranged legion or a portion thereof, each nested setbeing capable of producing a plurality of sequence reads each extendingin the same direction and each starting from a different position on thenucleic acid from which the nested set was generated: (c) sequencingsaid spatially isolated individual molecules; and (d) determiningabundances of different sequences of the nucleic acid molecules from thenucleic acid sample to generate the clonotype profile. In oneembodiment, the step of sequencing includes producing a plurality ofsequence reads for each of the nested sets. In another embodiment, eachof the somatically rearranged regions comprise as V region and a Jregion, and each of the plurality of sequence reads starts from adifferent position in the V region and extends in the direction of itsassociated J region.

In one aspect, for each sample from an individual, the sequencingtechnique used in the methods of the invention generates sequences ofleast 1000 clonotypes per run; in another aspect, such techniquegenerates Sequences of at least 10,000 clonotypes per run; in anotheraspect, such technique generates sequences of at least 100,000clonotypes per run; in another aspect, such technique generatessequences of at least 500,000 clonotypes per run; and in another aspect,such technique generates sequences of at least 1,000,000 clonotypes perrun. In still another aspect, such technique generates sequences ofbetween 100,000 to 1,000000 clonotypes per run per individual sample. Ineach of the foregoing, each clonotype per run is determined from atleast 10 sequence reads.

The sequencing technique used in the methods of the provided inventioncan generate about 30 bp, about 40 bp, about 50 bp, about 60 bp, about70 bp, about 80 bp, about 90 bp, about 100 bp, about 110, about 120 byper read, about 150 bp, about 200 bp, about 250 bp, about 300 bp about350 bp, about 400 bp, about 450 bp, about 500 bp, about 550 bp, or about600 by per read.

Clonotype Determination from Sequence Data

In some embodiments of the invention, sequence tags are used todetermine clonotypes and in other embodiments, sequence tags incombination with a sequence read coalescing step are used to determineclonotypes in embodiments in which a single unique sequence tag isattached to substantially every distinct target polynucleotide,clonotype determination using sequence tags is straight forward. In suchembodiments, clonotypes of a sample are determined by fast groupingsequence reads based on their sequence tags. Such grouping may beaccomplished by conventional sequence alignment methods. Guidance forselecting alignment methods is available in Batzoglou. Briefings inBioinformatics, 6: 6-22 (2005), which is incorporated by reference.After sequence reads are assembled. In groups corresponding to uniquesequence tags, then the sequences of the associated clonotypes may beanalyzed to determine the sequence of the clonotype front the sample.FIG. 4 illustrates an exemplary alignment and method from determiningthe sequence (SEQ ID NO: 2) of a clonotype associated with a uniquesequence tag. In this example, eleven sequence reads are aligned by wayof their respective sequence tags (4302) after which nucleotides at eachposition of the clonotype portions (4304) of the sequence reads,indicated as 1, 2, 3, 4, . . . n, are compared. For example, nucleotidesat position 6 (4306) are t, t, g, t, t, t, t, t, t, c, t: that is, ninebase calls are t's, one is “g” (4308) and one is “c” (4310) (SEQ ID NO:3 and SEQ ID NO: 4). In one embodiment, the correct base call of theclonotype sequence at a position is whatever the identity of themajority base is. In the example of position 6 (4306), the base call is“t”, because it is the nucleotide in the majority of sequence reads atthat position. In other embodiments, other factors may be taken intoaccount to determine a correct base call for a clonotype sequence, suchas quality scores of the base calls of the sequence reads, identities ofadjacent bases, or the like. Once clonotypes are determined as describedabove, a clonotype profile comprising the abundances or frequencies ofeach different clonotype of a sample may be assembled.

In some embodiments, more than one extension step may be carried outusing sequence-tag containing primers in order to increase the fractionof target polynucleotides in a sample that are labeled With sequencetags prior to amplification. In such embodiments, the more than oneextension steps in the presence of sequence tag-containing primersresults in a target polynucleotide and/or its copies being labeled witha plurality of different sequence tags. The size of the pluralitydepends on the number of extension steps carried out in the presence ofthe sequence tag-containing primers, the efficiency of the amplificationreaction, whether only one or both the forward and reverse primers havesequence tags, and the like. In some such embodiments, the plurality isin the range of from 2 to 5, or in the range of from 2 to 10, or in therange of from 2 to 5. In some such embodiments, after amplification,copies of each target polynucleotide of a sample may be divided into aplurality of groups or subsets wherein members of each group or subsetis labeled with the same sequence tag and members of each differentgroup or subset is labeled with a different sequence tag; that is,members of the same group have the same sequence tag and members ofdifferent groups hove different sequence tags. In other words, afteramplification, in some embodiments, each copy of a target polynucleotidefrom a sample will be labeled with one or two different sequence tags;or in other embodiments, each copy of a target polynucleotide from asample will be labeled with one of three different sequence tags; or inother embodiments, copies of a target polynucleotide from a sample willbe labeled with one of four different sequence tags; and so on. In thoseembodiments, clonotypes may be determined by a combination of sequencetag alignment followed by coalescing steps for treating sequence readswithin a group as originating from the same parent sequence based on alikelihood that common origin is true as a function of error rates,relative frequencies, and the like. FIG. 4B illustrates sequence readsfrom such an embodiment. In one approach, sequence roads are firstgrouped by common sequence tags (4402), which in the illustrationresults in three groups (4420), (4422) and (4424). In some embodiments,within each group, sequences (4404) are analyzed to determine aconsensus sequence of the group; for example, as above, at eachnucleotide position a base may be called as the majority base, or thehighest frequency base, or the like. The group consensus sequences maythen be coalesced with one another to determine clonotypes.

In some embodiments, the above aspect of the invention may beimplemented in a method for profiling of virtually any population ofnucleic acids in a sample. Such method may comprise the steps: (a)obtaining a sample comprising a population of nucleic acids; (b)attaching sequence tags to nucleic acids of the population to formtag-nucleic acid conjugates, wherein at least one nucleic acid of thepopulation or copies thereof have different sequence tags attached; (c)amplifying the tag-nucleic acid conjugates; (d) sequencing thetag-nucleic acid conjugates to generate sequence reads having errorrates and comprising a nucleic acid sequence and a tag sequence; (e)aligning sequence reads having like tag sequences to form groups ofsequence reads having the same sequence tags; (f) coalescing sequencereads of groups to determine sequences of the nucleic acids, whereingroups of sequence reads are coalesced into different sequences wheneversaid groups of sequence reads are distinct with a likelihood of at leastninety-five percent; and. (g) determining the sequence profile of thepopulation by determining the levels of the sequences. As applied toprofiling a population of recombined nucleic acids, such method may beimplemented by the steps: (al obtaining a sample from an individualcomprising T-cells and/or B-cells and/or cell-free DNA; (b) attachingsequence tags to recombined nucleic acid molecules of T-cell receptorgenes or immunoglobulin genes front the sample to form tag-nucleic acidconjugates, wherein at least one recombined nucleic acid from the sampleor copies thereof have different sequence lags attached; (c) amplifyingthe tag-nucleic acid conjugates: (d) sequencing the tag-nucleic acidconjugates to provide sequence reads having error rates and comprising atag sequence and a recombined nucleic acid sequence; (e) aligningsequence reads having like tag sequences to form groups of sequencereads having the same sequence tags: (f) coalescing sequence reads ofgroups to determine clonotypes, wherein groups of sequence reads arecoalesced into different sequences whenever said groups of sequencereads are distinct with a likelihood of at least ninety-five percent;and (g) determining the clonotype profile of the sample by determininglevels of the clonotypes.

In the above embodiments and other embodiments disclosed herein, thestep of sequencing tag-nucleic acid conjugates comprises sequencing asample of tag-nucleic acid conjugates from amplicon. Usually, suchsample is a representative sample in that the relative frequencies ofthe target polynucleotides in the original sample (that is, the tissuesample, blood sample, or the like) are maintained in the sample oftag-nucleic acid conjugates from the product of an amplificationreaction. In some embodiments in which population of recombined nucleicacids encoding immune receptor molecules are analyzed, a sample oftag-nucleic acid conjugates comprises at least 10⁴ tag-nucleic acidconjugates; in other embodiments, such sample comprises at least 10⁵tag-nucleic acid conjugates; in other embodiments, such sample comprisesal least 10⁶ tag-nucleic acid conjugates: in other embodiments, suchsample comprises at least 10⁷ tag-nuclei acid conjugates.

Coalescing Sequence Reads

In embodiments where multiple sequence tags are attached to an originalrecombined nucleic acid or copies thereof, a step of coalescing sequencereads (or consensus sequence reads from groups) may be carried out fordetermining clonotypes. Reducing a set of sequence reads for a givensample to a set of distinct clonotypes and recording the number of readsfor each clonotype would be a trivial if sequencing technology was errorfree. However, in the presence of sequencing errors, each genuineclonotype is surrounded by a ‘cloud’ of sequence reads with varyingnumbers of errors with respect to the its sequence. The “cloud” osequencing errors drops off in density as the distance increases fromthe clonotype in sequence space. A variety of algorithms are availablefor converting sequence reads into clonotypes. In one aspect, coalescingof sequence reads t that is, merging candidate clonotypes determined tohave one or more sequencing errors) depends on at least three factors;the number of sequences obtained for each of the clonotypes beingcompared; the number of bases at winch they differ; mid the sequencingquality score at the positions at which they are discordant. In someembodiments, a likelihood ratio may be constructed and assessed that isbased on the expected error rates and a binomial distribution oferrors.. For example, two clonotypes, one with 150 reads and the otherwith 2 reads with one difference between them in an area of poorsequencing quality will likely be coalesced as they are likely to begenerated by sequencing error. On the other hand two clonotypes, onewith 100 reads and the other with 50 reads with two differences betweenthem are not coalesced as they are considered to be unlikely to begenerated by sequencing error. In some embodiments, the algorithmdescribed below may be used for determining clonotypes from sequencereads. Sonic of these concepts are illustrated in FIG. 5A. In someembodiments of a coalescing step, sequence reads are first convertedinto candidate clonotypes. Such a conversion depends on the sequencingplatform employed. For platforms that generate high Q score longsequence reads, the sequence read or a portion thereof may be takendirectly as a candidate clonotype. For platforms that generate lower Qscore shorter sequence reads, some alignment and assembly steps may berequired converting a set of related sequence reads into a candidateclonotype. For example, for Solexa-based platforms, in some embodiments,candidate clonotypes are generated from collections of paired reads frommultiple clusters, e.g., 10 or more, as mentioned above.

The frequencies of candidate clonotypes may be plotted in sequencespace, as illustrated in FIG. 5A, where such space is reduced to onedimension (the horizontal axis) for sake of illustration. The verticalaxis gives the magnitude of each candidate clonotype's frequency,log(read count), or some like measure. In the figure, candidateclonotypes are represented by the various symbols (530). In accordancewith one embodiment of the invention, whether two candidate clonotypesare coalesced depends on their respective frequencies or read counts (asnoted above), the number of base differences between them (the moredifferences, the less likely is coalescence), and the quality scores ofthe bases at the locations where the respective sequences differ (higherquality scores makes coalescence less likely). Candidate clonotypes maybe considered in the order of their respective frequencies. FIG. 5Ashows candidate clonotype 1 (532), candidate clonotype 7 (534) andcandidate clonotype 11 (536) as the three candidates with the highestthree frequencies. Related to each such candidate clonotype are othercandidate clonotypes that are close in sequence, but with lesserfrequencies, such as (i) for candidate clonotype 1 (532) there arecandidate (Autotype 2 (538) and the candidate clonotypes 3, 4, 5 and 6enclosed by cone (540); for candidate clonotype 7 (534) there arecandidate clonotypes 8, 9 and 10 enclosed by cone (542); and (iii) forcandidate clonotype 11, there is candidate clonotype 12 enclosed by cone(544). The cones represent likelihood boundaries within which a lesserfrequency candidate clonotype would he coalesced with one of the higherfrequency candidate clonotypes 1, 7 or 11. Such likelihood boundariesare functions of the frequency of the nearby candidate clonotypes (3, 4,5 and 6 for 1; 8, 9 and 10 for 7; and 12 for 11) and their distances insequence space from the respective higher frequency candidateclonotypes. Candidate clonotype 2 (538) is outside cone (540); thus, itwould not be coalesced with candidate clonotype 1 (532). Again, thelikelihood (of coalesce) boundaries are shown as cones because candidateclones with higher frequencies are more likely to be genuinely differentclonotypes than those of lower frequencies and multiple differences atlower frequencies are more likely to be errors than multiple differencesat higher frequencies.

The cloud of sequence reads surrounding, each candidate clonotype can bemodeled using the binomial distribution and a simple model for theprobability of a single base error. This latter error model can beinferred from mapping V and J segments or from the clonotype findingalgorithm itself, via self-consistency and convergence. A model isconstructed for the probability of a given ‘cloud’ sequence Y with readcount C2 and E errors (with respect to sequence X) being' art of a trueclonotype sequence X with perfect read count C1 under the null modelthat X is the only true clonotype in this region of sequence space. Adecision is made whether or not to coalesce sequence Y into theclonotype X according the parameters C1, C2, and E. For any given C1 andE a max value C2 is pre-calculated for deciding to coalesce the sequenceY. The max values for C2 are chosen so that the probability of failingto coalesce Y under the null hypothesis that Y is part of clonotype X isless than some value P after integrating over all possible sequences Ywith error E in the neighborhood of sequence X. The value P is controlsthe behavior of the algorithm and makes the coalescing more or lesspermissive.

If a sequence Y is not coalesced into clonotype X because its read countis above the threshold C2 for coalescing into clonotype X then itbecomes a candidate for seeding separate clonotypes (such as withcandidate clonotype 2 (538) in FIG. 5A). An algorithm implementing suchprinciples would also make sure that any other sequences Y2, Y3, etc.which are ‘nearer’ to this sequence Y (that had been deemed independentof X) are not aggregated into X. This concept of ‘nearness’ includesboth error counts with respect to Y and X and the absolute read count ofX and Y, i.e. it is modeled in the same fashion as the above model forthe cloud of error sequences around clonotype X. In this way ‘cloud’sequences can be properly attributed to their correct clonotype theyhappen to be ‘near’ inure than one clonotype. Thus, going to FIG. 5A, itcandidate clonotype 2 is deemed to be genuinely distinct from candidateclonotype 1 (532), then a special routine, or sub algorithm, wouldprovide a rule for determining which of candidate clonotypes 1 (532) and2 (538), candidates 4 and 5, between 1 and 2, should be coalesced to (ifeither).

In one embodiment, an algorithm proceeds in a top down fashion bystarting with the sequence X with the highest read count. This sequenceseeds the first clonotype. Neighboring sequences are either coalescedinto this clonotype if their counts are below the pre calculatedthresholds (see above), or left alone if they are above the threshold or‘closer’ to another sequence that was not coalesced. After searching allneighboring sequences within a maximum error count, the process ofcoalescing reads into clonotype X is finished, its reads and all readsthat have been coalesced into it are accounted for and removed front thelist of reads available for making other clonotypes. The next sequenceis then moved on to with the highest read count. Neighboring reads arecoalesced into this clonotype as above and tins process is continueduntil there are no more sequences with read counts above a giventhreshold, e.g. until all sequences with more than 1 count have beenused as seeds for clonotypes.

As mentioned above, in another embodiment of the above algorithm, afurther test may be added for determining whether to coalesce acandidate sequence Y into an existing clonotype X, which takes intoaccount quality score of the relevant sequence reads. The averagequality score(s) are determined for sequence(s) Y (averaged across allreads with sequence Y) were sequences Y and X differ. If the averagescore is above a predetermined value then it is more likely that thedifference indicates a truly different clonotype that should not becoalesced and if the average score is below such predetermined valuethen it is more likely that sequence Y is caused by sequencing errorsand therefore should be coalesced into X.

Successful implementation of the above algorithm for coalescingcandidate clonotypes is dependent upon having an efficient way offinding all sequences with less than E errors (i.e. less than somesequence distance measure) from some input sequence X. This problem maybe solved using it sequence tree. The implementation of such trees hassome unusual features in that the nodes of the tree are not restrictedto being single letters of the DNA sequences of the candidateclonotypes, as illustrated in FIG. 5D. The nodes can have arbitrarilylong sequences, which allows for a more efficient use of computermemory.

All of the reads of a given sample are placed into the sequence tree.Each leaf nodes holds pointers to its associated reads. A uniquesequence of a candidate clonotype retrieved by traversing backwards inthe tree front the leaf to the root node. The first sequence is placedinto a simple tree with one root node and one leaf node that containsthe full sequence of the read. Sequences are next added one by one. Foreach added sequence either a new branch is formed at the last point orcommon sequence between the read and the existing tree or add the readto an existing leaf node if the tree already contains the sequence.Having placed all the reads into the tree it is easy to use the tree forthe following purposes: 1) Finding the highest read count: soiling leafnodes by read count allows one to hind the leaf node (i.e. sequence)with the most reads, and successively lower numbers of reads; 2) Findingneighboring leafs: for any sequence all paths through the tree whichhave less than X errors with respect to this sequence are searehable. Apath is started at the root and branch this path into separate pathsproceeding along the tree. The current error count of each path asproceeding along the tree is noted. When the error count exceeds the maxallowed errors the given path is terminated. In this way large parts ofthe tree are pruned as early as possible. This is an efficient way offinding all paths (i.e. all leafs) within X errors from any givensequence.

Features of the above concepts are illustrated in more detail in theflow chart of FIG. 5B. A set of candidate clonotypes is obtained fromsequence data obtained by sequencing recombined nucleic acids extractedfrom a sample of T cells or B cells. In one aspect, candidate clonotypeseach include an NDN region and portions of V and J regions. Thesesequences are organized into a data structure (550). Which may be asequence tree. Not shown in FIG. 5B, as part of generating a set ofcandidate clonotypes, in one embodiment, sequence trees may also beconstructed for known V regions and known J regions. Sequence readsmaking up a candidate clonotype may then be mapped, or aligned, to theseknown sequences via the sequence trees to efficiently determine the mostlikely known V and J sequences of the candidate clonotypes. Retaining toFIG. 5B, once the candidate clonotypes are generated, a data structure,such as a sequence tree, is constructed for use in a method fordistinguishing genuine clonotypes front candidate clonotypes thatcontain experimental or measurement errors, such as sequencing errors.The candidate clonotype that has the highest frequency of occurrenceamong the current candidate clonotypes (HFCC_(k)) is selected (552) fromthe data structure, for example a sequence tree: in other words,HFCC_(k) is the candidate clonotype with the highest number occupies, orread counts in cycle k. Next, neighboring lesser frequency candidateclonotypes are identified (LFCCs) (554); that is, candidate clonotypeswithin a distance of D_(k) are identified. In one aspect of theinvention, this identification is carried out using a sequence treewhich allows efficient sequence comparisons relatively short (<300 bp)sequences.

In one embodiment, the comparisons, or sequence alignments, are carriedout using dynamic programming, e.g. as disclosed by Gusfield (editedabove.). In a further embodiment, such dynamic programming is bandeddynamic programming where sequences that differ from the selected HFCCby more than a predetermined distance are not considered, which speedsthe computation. The candidates HFCC_(k) and LFCC_(j) may be compared onthe basis of many different criteria or properties. In one aspect, asmentioned above, candidate clonotypes are compared on the basis of atleast two properties, (i) frequency or read counts and (ii) sequencedifferences. In another aspect, as mentioned above, candidate clonotypesare compared. On the basis of at least three properties: (i) frequencyor read counts, (ii) sequence differences, and (iii) quality scores ormeasures of the bases where differences occur. In a one embodiment,sequence differences include base substitution; in another embodiment,sequence differences include base substitutions, deletions andinsertions. The latter embodiment is especially applicable wheneversequence data is generated by sequencing-by-synthesis chemistries thatdo not employ terminators, such as 454 sequencers and Ion Torrentsequencers. Such sequencing approaches differentiate different sizedhomopolymer stretches by signal amplitude; thus, base-calling routinesin such approaches are prone to insertion and deletion errors, becausethe difference in signal level from homopolymers differing by onenucleotide drops precipitously with increasing homopolymer size (thatis, a 2-mer is readily distinguished from a 3-mer, but an 8-mer isalmost indistinguishable from a 9-mer). In one aspect, comparisons ofHFCCs and LFCCs may be implemented using a function (referred to hereinas a “coalescence likelihood function”), such as P(HFCC_(k), LFCC_(j),D, Q) shown in decision box (558), which depends on the quantities (i)through (iii) described above. Such a function may take many diluentforms, but generally the value of P changes with changes in (i), (ii)and (iii) as follows: The value of P preferably increases monotonicallywith the frequency of HFCC and the ratio of the frequency of HFCC tothat of LFCC, such that the higher the ratio of the frequency of HFCC tothat of LFCC, the higher the likelihood LFCC will be coalesced intoHFCC. Likewise, the value of P preferably decreases monotonically withdegree to which the sequences of HFCC and LFCC differ, so that thegreater the difference between HFCC and LFCC (e.g. as measured by theminimal number of substitutions, insertions or deletions to change oneto the other) the lower the likelihood LFCC will be coalesced with HFCC.Finally, the value of P preferably decreases monotonically withincreasing quality scores of the locations where the sequences of HFCCand LFCC differ, so that for higher quality scores, the lower thelikelihood LFCC will be coalesced with HFCC.

When the sequences of HFCC and LFCC differ at more than one location,the quality scores a different locations may be combined in a variety ofdiffer ways. In one embodiment, whenever there is a plurality of suchdifferences, the plurality of quality scores is expressed as an averagevalue, which may be either an unweighted average or a weighted average.FIG. 5C shows an exemplary function, P, computed for different qualityvalues (curves a through c) for a given sequence difference. Asillustrated in FIG. 5C, whenever HFCC is at a level of about 200 readcounts (570), then if the quality scores are determined by curve (a),any LFCC with less than about 50 read counts (572) are coalesced intoHFCC. The argument, D, of function P is a measure of the distancebetween the sequences HFCC_(k) and LFCC_(j) and its value may vary fromcycle to cycle as an analysis progresses. (The indices “k” indicatesthat the values of constants with a “k” subscript may depend on thecomputational cycle, k.) In one embodiment, D=D_(k), so that its valueis a function of cycle number. In another embodiment, D=D(HFCCfrequency), so that its value is a function of the frequency of HFCC,independent of cycle number. For example, as the frequency of HFCCdecreases, then distance, D, of candidates to be compared decreases. Inone embodiment, D is it Hamming distance between HFCC_(k) and LFCC_(j);however, other distance measures may be used. In one embodiment, D_(k)is a non-increasing function of k; and in another embodiment, D_(k) is adecreasing function of k. Decreasing the magnitude of D with increasingcycle number, or with decreasing frequency of HFCC, is advantageous insome embodiments because as a computation progresses to lower and lowerfrequency candidate clonotypes most such candidates are singletons, sothat sequence distance (rather than frequency difference) becomes thepredominant comparison. By lowering D as the computation progresses,unproductive comparisons to distant low frequency candidate clonotypesare reduced, thereby speeding up the computation. Function P may be acomplicated expression depending on the number of factors beingconsidered. FIG. 5C illustrates computed values for one embodiment of Pwhich relates read count thresholds for coalescing an LFCC given a readcount of an HFCC for different quality scores, as described above.Curves “a” through “e” represent the relationships for different qualityscores (with curve “a” corresponding to the highest quality score).

Returning to FIG. 5B, if P<P_(k), then LFCC_(j) is not coalesced withHFCC_(k) and another LFCC is selected (560), if P>P_(k), then LFCC_(j)is coalesced with HFCC_(k) (562), in which case another LFCC is selected(566), unless there are no more LFCC left to evaluate (564). If thereare no more LFCC to evaluate (564), then the current HFCC_(k) (includingall of the LFCC's coalesced into it) is removed (518) from the datastructure, such as the sequence tree. Such removal is illustrated in thesimple sequence tree (590) of FIGS.5D-5E. There, path (592) (indicatedby dashed line) in sequence tree (590) corresponds to HFCC (596), whichis coalesced with LFCC (598). After coalescence, the segment of path(592) in shaded area (599) is removed from sequence tree (590) to givereduced sequence tree (597) shown in FIG. 5E, which is used insubsequent computations to find neighboring LFCC (554). After suchremoval, clonotype determination is finished if a stopping criterion(570) is met. In one embodiment, stopping criterion (570) is whether thelast non-singleton candidate clonotype has been processed (552). Inanother embodiment, stopping criterion (570) is whether the frequency orthe read counts of the selected HFCC is below that corresponding to asingle lymphocyte. In one aspect of the method of the invention, anamplification step may result in each lymphocyte in a sample beingrepresented by multiple copies of the same clonotype; thus, in oneembodiment, whatever HFCC has a number of read counts below the numbercorresponding to a single lymphocyte, then the computation is stopped.In some embodiments, such a number of read counts (or candidateclonotype copies) is at least 10; in another embodiment, such number isat least 20; in another embodiment, such a number is at least 30; inanother embodiment, such a number is at least 40. If the stoppingcriterion is not met, then the next HFCC is selected (572). Theanalytical steps summarized in the flow chart of FIG. 5B may beimplemented in any suitable programming language, such as C, C++, Java,C#, Fortran, Pascal or the like.

In accordance with one aspect of the invention, the above method fordetermining clonotypes and/or clonotype profiles comprises steps of (a)forming a data structure of recombined immune molecules front sequencereads obtained by high throughput nucleic acid sequencing, (b)coalescing with a highest frequency candidate clonotype any lesserfrequency candidate clonotypes whenever such lesser frequency is belowit predetermined frequency value and a sequence difference therebetweenis below it predetermined difference value to form a clonotype, (c)removing the coalesced candidate clonotype from the data structure, and(d) repeating steps (b) and (c) until a clonotype profile is formed. Inone embodiment, the data structure is a sequence tree.

In accordance with another aspect of the invention, the above method ofdetermining clonotypes may be carried out by steps comprising: (a)providing a set of sequence reads from a repertoire of recombined immunemolecules each having a V region, an NDN region and a J region whereinfor each such molecule at least one sequence read encompasses at least aportion of the NDN region of such molecule; (b) farming from sequencereads encompassing at least a portion of an NDN region a sequence treehaving leaves representing candidate clonotypes, each leaf and itscorresponding candidate clonotype having a frequency: (c) coalescingwith a highest frequency candidate clonotype any lesser frequencycandidate clonotypes whenever such lesser frequency is below apredetermined frequency value and a sequence difference therebetween isbelow a predetermined difference value to form a clonotype having asequence of the highest frequency candidate clonotype; (d) removingleaves corresponding to the coalesced candidate clonotypes from thesequence tree; and (e) repeating steps (c) and (d) until a highestfrequency of a lesser frequency candidate clonotype is below apredetermined stopping value. In one embodiment, the step of formingfurther includes selecting a highest frequency candidate clonotype andidentifying all said lesser frequency candidate clonotypes having asequence difference therewith less than a predetermined difference valueto form a coalescence subset. Thus, in such embodiment, one may limitthe total number of LFCCs that must be compared for the coalescingoperation (only ones within the predetermined difference value areconsidered). Such value is a process input depending on the application,e.g. the size of the repertoire, how much computing binge is used, andso on. As mentioned above, the function used for deciding whether tocoalesce an HFCC with a LFCC can have a variety of forms. In one generalaspect, for the step of coalescing, such a function may have thefollowing properties. It depends on frequencies of HFCC, LFCC, thesequence difference therebetween (which may be expressed as aconventional string difference measure, such as a Hamming distance) andquality scores of the one or more nucleotide locations where the HFCCand LFCC differ; such that the function (i) monotonically increases withincreasing ratio of frequency of HFCC and frequency of LFCC, (ii)monotonically decreases with increasing sequence difference between HFCCand LFCC, and (iii) monotonically decreases with increasing qualityscores of the one or mare nucleotide locations. That is, in regard toproperty (iii), the surer one is that HFCC and LFCC are different (e.g.,because there is a high level of confidence in the base calls), then theless likely they will be coalescenced.

In some embodiments, a coalescence likelihood function is selected sothat sequence reads are coalesced into different clonotypes (or targetpolynucleotides, such as recombined nucleic acids) whenever suchsequence reads are distinct with a likelihood or at least 95 percent; inother embodiments, a coalescence likelihood function is selected so thatsequence reads are coalesced into different clonotypes whenever suchsequence reads are distinct with a likelihood of at least 99 percent; inother embodiments, a coalescence likelihood function selected so thatsequence reads are coalesced into different clonotypes whenever suchsequence reads are distinct with a likelihood of at least 99.9 percent.As mentioned above, in some embodiments, a coalescence likelihoodfunction depends on an error rate of a sequencing chemistry used, theamber of discrepant nucleotides in sequence reads being compared, andthe relative frequencies of the sequence reads being compared; inanother embodiment, a coalescence likelihood function depends on anerror rate of a sequencing chemistry used., the number of discrepantnucleotides in sequence reads being compared, the relative frequenciesof the sequence reads being compared, and the quality scores of thediscrepant nucleotides. In the foregoing, selection of a predeterminedfrequency value and a predetermined difference value is a design choicethat depend on particular applications. Factors affecting such choicesmay include details of the biology, speed of implementation, and thelike.

Monitoring Applications

In one aspect, the invention is directed to methods for monitoringminimal residual disease by determining the presence, absence and/orlevel of nucleic acids in a sample that are characteristic or correlatedwith a disease. In some embodiments, such nucleic acids are somaticallyrecombined nucleic acids, or clonotypes, which are correlated with apre-cancerous or cancerous condition, such as a lymphoid or myeloidproliferative disorder, and which can be used to monitor the status ofthe disorder or condition. Such nucleic acids, and in particularclonotypes, are useful for monitoring minimal residual disease of acancer after treatment, where the result of such monitoring is a keyfactor in determining whether to continue, discontinue or otherwisemodify treatment. In many malignant lymphoid and myeloid neoplasms, adiagnostic tissue sample, such as a peripheral blood sample or a bonemarrow sample, is obtained before treatment from which a clonotypeprofile is generated (a “diagnostic clonotype profile”). For lymphoid ormyeloid proliferative disorders, it is usually not known prior to adiagnostic sample which immune receptor chain(s) are correlated to thelymphoid or myeloid clone of the disorder or condition. Consequently,under current practice many separate amplifications and sequencing mustbe carried out on different recombined nucleic acids encoding differentcandidate immune receptor chains in order to identify clonotypescorrelated with a patient's disease or condition. One or moredisease-correlated clonotypes (i.e. “correlating clonotypes”)areidentified in clonotype profiles resulting from such amplifications andsequencing efforts. Typically, the clonotypes having the highestfrequencies in the clonotype profiles are taken as the correlatingclonotypes. In one aspect of the invention, the number of separateamplifications and sequencing runs necessary to identify correlatingclonotypes is greatly reduced by providing larger scale multiplexamplifications in a single reaction of portions of recombined nucleicacids encoding a plurality of difference immune receptor chains. In someembodiments, such plurality is in the range of from 2 to 4 separateimmune receptor chains; and in other embodiments, such plurality is inthe range of from 2 to 3 separate immune receptor chains. Moreparticularly, in some embodiments, among BCR chains the following areamplified in a single multiplex reaction: recombined nucleic acidsencoding IgH including at least a portion of the VDJ region, IgHincluding at least a portion of the DJ region, and IgK; and in otherembodiments, among TCR chains the following are amplified in a singlemultiplex reaction: TCRβ, TCRδ and TCRγ.

After treatment, and preferably after attainment of as completeremission of the cancer, the presence, absence or frequency of suchcorrelating clonotypes for nucleic acids is assessed periodically todetermine whether the remission is holding or whether the neoplasm isreturning or relapsing, based on the presence of, or an increase in thefrequency of, the correlating nucleic acids or clonotypes (or relatedclonotypes) in a post-treatment clonotype profile or nucleic acidprofile. That is, after treatment, minimal residual disease of thecancer is assessed based on the presence, absence or frequency of thecorrelating clonotypes or characteristic nucleic acids. As mentionedabove, when such correlating clonotypes are common or correspond to arearranged receptor segment that lacks sufficient diversity (so thatnon-cancerous cells may share the clonotype), the occurrence of suchclonotypes in a post-treatment clonotype profile may give rise to afalse positive indication of relapse.

Methods of the invention are applicable to monitoring any proliferativedisease in which a rearranged nucleic acid encoding an immune receptoror portion thereof can be used as a marker of cells involved in thedisease. In one aspect, methods of the invention are applicable tolymphoid and myeloid proliferative disorders. In another aspect, methodsof the invention are applicable to lymphomas and leukemias. In anotheraspect, methods of the invention are applicable to monitoring MRD infollicular lymphoma, chronic lymphocyte leukemia (CLL), acute lymphocyteleukemia (ALL), chronic myclogenous leukemia (CML), acute myelogenousleukemia (AML), Plodgkins's and non-Hodgkin's multiple myeloma (MM),monoclonal gammopathy of undetermined significance (MGUS), mantle celllymphoma (MCL), diffuse large B cell lymphoma (DLBCL), myelodysplasticsyndromes (MDS), T cell lymphoma, or the like. In a particularembodiment, a method of the invention is particularly suited formonitoring MRD in ALL, MM or DLRCL.

In some embodiments, a patient sample, such as blood or bone marrow, issubjected to a diagnostic assay to identify which of a plurality ofimmune receptor chains may include the clonotype produced by a clone ofa disorder (i.e., a correlating clonotype). Once the immune receptorchain of a correlating clonotype is determined, then subsequentmonitoring assays may be specific for that particular immune receptorchain. For example, in some embodiments, a diagnostic assay may in thesame reaction generate sequence-based clonotype profiles of a pluralityof BCR chains, such as, IgH(VDJ), IgH(DJ) and IgK. If a correlatingclonotype is an IgH(VDJ) chain, then subsequent monitoring assays mayonly generate IgH(VDJ) clonotype profiles. In some embodiments, thedepth of sequencing in the diagnostic sample may be different than thatof the monitoring sample. “Depth o f sequencing” means the total numberof sequence reads analyzed to construct clonotype profiles. For cancers,such as leukemias or lymphomas, since diagnostic assays are conducted onpatient samples prior to treatment, the frequeney or level of acorrelating clonotype in the sample is typically high and readilyidentified, For example, any clonotype with a frequency over apredetermined level may be defined as a correlating clonotype. Suchpredetermined level may vary under with other patient indicators;however, often a predetermined level may be in the range of from 2 to 5percent; or in some embodiments, five percent. Thus, in someembodiments. the depth of sequencing carried out is that which isnecessary to reliably detect clonotypes present at a frequency of one ortwo percent or higher. In some embodiments, the depth of sequencing of adiagnostic sample produces at least 10,000 sequence reads; or in otherembodiments, it is at least 100,000 sequence reads; in still otherembodiments, the depth of sequencing of a diagnostic sample produces atleast 10⁶ sequence reads. In some embodiments, the depth of sequencingor a monitoring sample is at least 10,000 sequence reads; in otherembodiments, the depth of sequencing of a monitoring sample is at least10⁸ sequence reads.

In some embodiments, a lymphoid proliferative disorder, such as aleukemia orlymphoma, in a patient may be monitored by generatingclonotype profiles from successively obtained samples (or tissuesamples) from the patient. Such clonotype profiles may be generated asdescribed above. In some embodiment, such monitoring may be implementedby the following steps: (a) obtaining a sample from an individualcomprising T-cells and/or B-cells and/or cell-free DNA; (b) attachingsequence tags to recombined nucleic acid molecules of T-cell receptorgenes or immunoglobulin genes from the sample to form tag-nucleic acidconjugates, wherein at least one recombined nucleic acid or copiesthereof have different sequence tags attached; (c) amplifying thetag-nucleic acid conjugates; (d) sequencing a sample of the tag-nucleicacid conjugates to provide sequence reads each having an error rate andeach comprising a tag sequence and a recombined nucleic acid sequence(e) aligning sequence reads having like tag sequences to form groups ofsequence reads having the sante sequence tags: (f) coalescing sequencereads of groups to determine clonotypes, wherein groups of sequencereads are coalesced into different recombined nuceie acid sequenceswhenever said groups of sequence reads are distinct with a likelihood ofat least ninety-five percent; (g) determining the clonotype profile ofthe sample by determining levels of the clonotypes; and (h) determiningthe level of correlating clonotypes in the clonotype profile. In someembodiments, steps (a) through (h) may be repeated in the process ofmonitoring the patient to determine whether the level of correlatingclonotypes is evidence of relapse of disease. In some embodiments, thesteps of attaching and amplifying may comprise the following steps: (a)combining in a reaction mixture under primer extension conditions afirst set of primers with a sample of recombined nucleic acids fromimmune cells expressing an immune receptor and/or cell-free DNA, whereineach primer of the first set has a receptor-specific portion such thatthe receptor-specific portion anneals to a different recombined nucleicacid at a predetermined location and is extended to form a firstextension product, and wherein each primer of the first set has a5′-non-complementary end containing a first primer binding site; (b)removing from the reaction mixture non-extended printers of the firstset; and (c) adding to the reaction mixture under primer extensionconditions a second set of primers, wherein each primer of the secondset has a receptor-specific portion such that the receptor-specificportion anneals to the first extension product at a predeterminedlocation and has a 5′-non-complementary end containing a second primerbinding site, primers of the first set and/or primers of the second setcomprising a sequence tag disposed between the receptor-specific portionand the first or second primer binding site, respectively, and whereineach primer of the second set is extended to form a second extensionproduct, such that each second extension product comprises a firstprimer binding site, a second primer binding site, at least one sequencetag, and recombined nucleic acid encoding a portion of an immune cellreceptor chain. In some embodiments, a step of coalescing recombinednucleic acids comprises coalescing sequence reads of differentrecombined nucleic acids whenever such sequence reads are distinct witha likelihood of at least ninety-nine percent; and in other embodiments,with a likelihood of at least 99.9 percent.

Methods of the invention are also applicable to monitoring minimalresidual disease of a cancer in a patient, including a non-lymphoid ornon-myeloid cancer which has an identifying pattern of mutations, forexample, in a selected set of cancer genes. Such a pattern of mutations,that is, the presence, absence and/or level of genes containing suchmutations, can indicate a likelihood of disease recurrence. In someembodiments, target polynucleotides for such monitoring may be exons,portions exons, selected introns and/or gene expression control regions,e,g. promoters, of a plurality of genes (referred to herein as “cancergene molecules”). Cancer gene molecules may be isolated from a tissuesample using conventional techniques, such as exon aperture techniques,e.g. TruSeq™ exon enrichment kit (Illumina, San Diego, Calif.); Framptonet al, Nature Biotechnology, 31(11): 1023-1031 (2013); and the like.After such cancer gene molecules are obtained, sequence tags areattached to form tag-nucleic acid conjugate, tag-nucleic acid conjugateare amplified and sequenced in accordance with the invention.

Recent cancer genome sequencing studies have shown that there issignificant heterogeneity in mutation patterns among different cancers,among different patients with the same cancer, among cells of the sametumor, and among cells of different metastatic sites in the samepatient; however, within the same patient, the heterogeneous cancercells typically evolve from a common ancestor, so that they sharemutations and the evolutionary relationship among the cancerous cellsmay be discerned in a succession of measurements over time, e.g.Vogelstein et al, Science, 339: 1546-1558 (2013):. Ding et al, Nature,481(7382): 506-510 (2012); and the like; therefore, a pattern ofmutations correlated with a cancer measured in a diagnostic sampleprovide a means to detect a recurrence of the same cancer or a clonallyevolved version of it.

Cancer gene molecule may be selected from a wide variety of genes,including, but not limited to, the genes in Table I.

TABLE I Exemplary Cancer Genes ABL1 AKT1 ALK APC ATM BRAF CDH1 CSF1RCTNNB1 EGFR ERBB2 ERBB4 FBXW7 FGFR1 FGFR2 FGFR3 FLT3 GNA11 GNAQ GNASHNF1A HRAS IDH1 JAK2 JAK3 KDR KIT KRAS MET MLH1 MPL NOTCH1 NPM1 NRASPGGFRA PIK3CA PTEN PTPN11 RB1 RET SMAD4 SMO SRC STK TP53 VHL

In some embodiments, the above method of monitoring a minimal residualdisease of a cancer may comprise the following steps: (a) obtaining froman individual a tissue sample; (b) attaching sequence tags to each of aplurality of cancer gene molecules in the sample to form tag-nucleicacid conjugates, wherein at least one nucleic acid or copies thereofhave different sequence tags attached and wherein the cancer genemolecules are characteristic of a cancer of the individual; (c)amplifying the tag-nucleic acid conjugates, (d) sequencing a sample ofthe tag-nucleic acid conjugates to provide sequence reads having errorrates and comprising a tag sequence and a cancer gene sequence; (e)aligning sequence reads having like tag sequences to form groups ofsequence reads having the same sequence tags, (f) coalescing cancer genesequences of groups to determine sequences of cancer gene molecules,wherein groups of sequence reads are coalesced into different cancergene molecules whenever said groups of cancer gene sequences aredistinct with a likelihood of at least ninety-five percent, and (g)detecting in a profile of the cancer gene molecules the presence,absence and/or level of cancer gene molecules characteristic of thecancer of the individual. In some embodiments, a step of coalescingcancer gene sequences comprises coalescing sequence reads of differentcancer gene molecules whenever such sequence reads are distinct with alikelihood of at least ninety-nine percent; and in other embodiments,with a likelihood of at least 99.9 percent.

Use of Sequence Tags to Detect Carry Over Contamination

Carry over contamination is a significant problem with techniques thatinclude amplification of nucleic acids, e,g. Borst et al, Eur. J. Clin.Microbial. Infect. Dis. 23(4): 289-299 (2004); Aslanzadeh, Ann. Clin.Lab. Sci. 34(4): 389-396 (2004); and the like. Such contamination ariseswhen traces of nucleic acid extraneous to a sample are unintentionallyamplified in an assay of the sample and effect or impact a measuredresult. In a worse case, carry over contamination in a medical samplefrom a patient can result in a false positive interpretation of an assayresult. The extraneous nucleic acid may conic from a source unrelated toa particular patient, for example, it may come from the sample ofanother patient. Or, the extraneous nucleic acid may come from a sourcerelated to a patient; for example, it may come from a different samplefrom the same patient handled in the same laboratory in the past or froman assay reaction on a different sample from the same patient which wasprocessed in the same laboratory in the past.

Carry over contamination is especially challenging in setting whenmeasuring highly complex populations of related nucleic acids, such aspopulations of recombined nucleic acids encoding immune molecules, suchas T-cell receptors or immunoglobulins. The challenge arises because itis difficult to determine whether a sequence read or clonotype is partof the genuine diversity of an intended sample or whether they originatefrom an extraneous source of nucleic acid, such as another patient'ssample or a prior sample of the same patient which are being processedin the same king of assay in the same laboratory. In one aspect of theinvention, such carry over contamination may be detected by usingsequence tags not only to determine clonotypes from sequence reads butalso to determine whether a sequence tag originated in the currentsample or from another sample. This is accomplished by maintaining arecord of sequence tags determined from each patient sample, thenwhenever a subsequent measurement is made the sequence tags of thecurrent measurement are compared to those of prior measurements. Suchrecords of sequence tags associated with clonotypes are convenientlymaintained as electronic records on mass storage devices because of thelarge number of tag from each measurement and the ease of searching andcomparing electronic records using conventional algorithms. If a matchis found then the most likely cause is carry over contamination,provided that the populations of sequence tags employed in themeasurements are sufficiently large. The same exemplary ratios of thesize of sequence tag population to a clonotype population for labelingby sampling discussed above are applicable for detecting carry overcontamination. In one embodiment, such ratio is 100:1 or greater.

A is variety of search methods or algorithms may be used to carry outthe step of comparing measured clonotypes to database clonotypes. Manyconventional sequence alignment and searching algorithms are publiclyavailable and have been described in the following references which areincorporated by reference: Mount, Bioinformatics Sequence and GenomeAnalysis, Second Edition (Cold Spring Harbor Press, 2004): Batioglou,Briefings in Bioinformatics, 6: 6-22 (2005); Altschul et al, J. Mol.Biol., 215(3): 403-410 (1990); Needletnan and Wunsch. J. Mol. Biol., 48:443-453 (1970); Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981): and the like.

In some embodiments, the above methods for detecting and measuringcontamination, such its carry-over contamination; in a sample frommaterial originating from a different sample may comprise the followingsteps: (a) obtaining from an individual a tissue sample, (b) attachingsequence tags to cancer gene molecules or recombined nucleic acids toform tag-nucleic acid conjugates, wherein at least one nucleic acid orcopies thereof have different sequence tags attached and wherein thecancer gene molecules are characteristic of a cancer of the iitdizideal: (c) amplifying the tag-nucleic acid conjugates: (d) sequencing asample of the tag-nucleic acid conjugates to provide sequence reads eachhaving an error rate and each comprising a lag sequence and a cancergene sequence or recombined nucleic acid sequence; (e) comparing tagsequences to separately determined tag sequences from other tissuesamples; and (f) determining the presence, absence rndbr level ofcontamination by the identity of one or more tag sequences with anyseparately determined tag sequences from other tissue samples. Once tagsequences are determined in an assay, they may be compared to tagsequences in a database of tag sequences recorded from assays on otherpatients. Such steps of comparing may be implemented at the time of anassay, or such steps may be implements retrospectively, for example, ata time after the time of the assay. In one embodiment, sequence tags areattached to recombined nucleic acids in a tissue sample, such, as bloodor bone marrow, front an individual suffering from a lymphoidproliferative disorder, such as a lymphoid cancer. In anotherembodiment, sequence tags are attached to cancer gene molecules, such asdescribed above.

In further embodiments in which recombined nucleic acids are monitoredform cross-contamination of tissue samples, the steps of attaching, andamplifying may be implemented as follows: (a) combining in a reactionmixture under primer extension conditions a first set of primers with asample of recombined nucleic acids form T-cells and/or cell-free DNA,wherein each primer of the first set has a receptor-specific portionsuch that the receptor-specific portion anneals to a differentrecombined nucleic acid at a predetermined location and is extended toform a first extension product, and wherein each primer of the first sethas a 5′-non-complementary end containing a first primer binding site;(b) removing from the reaction mixture non-extended primers of the firstset: (e) adding to the reaction mixture under primer extensionconditions a second set of primers, wherein each primer of the secondset has a receptor-specific portion such that the receptor-specificportion anneals to the first extension product at a predeterminedlocation and has a 5′-non-complementary end containing a second printerbinding site, primers of the first set and/or primers of the second setcomprising a sequence tag disposed between the receptor-specific portionand the that or second primer binding site, respectively, and whereineach primer of the second set is extended to form a second extensionproduct, such that each second extension product comprises a thatprinter binding site, a second primer binding site, at least onesequence tag, and recombined nucleic acid encoding a portion of immunereceptor chain: and (d) performing a polymerase chain reaction in thereaction mixture to form an amplicon, the polymerase chain reactionusing forward primers specific for the first primer binding site andreverse primers specific for the second primer binding site.

Kits

The invention includes a variety of kits for carrying out methods of theinvention. In some embodiments, kits comprise (a) a set of forwardprimers and a set of reverse primers for amplifying in a multiplex PCRrecombined nucleic acids encoding a plurality of immune receptor chainswherein forward primers and: or reverse primers each have a targetspecific portion, a sequence tag and a common primer binding site, and(b) a primer removal element for removing after at least a firstextension unincorporated primers non-extended primers) of the sets someembodiments, kits further comprise common primers specific for thecommon printer binding sites. In some embodiments, kits thrillercomprise written instructions for using in components in a method of theinvention. In some embodiments, kits further comprise forward andreverse primers specific for amplifying recombined nucleic acidsencoding IgH(VDJ), IgH(DJ) and IgK. In some embodiments, kits furthercomprise forward and reverse primers specific for amplifying recombinednucleic acids encoding TCRβ, TCRδ and TCRγ. In some embodiments, kitsfurther comprise internal standards comprising a plurality of nucleicacids having lengths and compositions representative of the targetrecombined nucleic acids, wherein the internal standards are provided inknown concentrations. In some embodiments, kits include asingle-stranded exonuclease as a printer removal element, such as E.coli exonuclease I. In some embodiments, kits include a spin columncapable of size selecting double stranded DNA as a primer removalelement,

While the present invention has been described with reference to severalparticular example embodiments, those skilled in the art will recognizethat many changes may be made thereto without departing from the spiritand scope of the present invention. The present invention is applicableto a variety of sensor implementations and other subject matter, inaddition to those discussed above.

Definitions

Unless otherwise specifically defined herein, terms and symbols ofnucleic acid chemistry, biochemistry, genetics, and molecular biologyused herein follow those of standard treatises and texts in the field,e.g. Kohlberg and Baker, DNA Replication: Second Edition (W.H., Freeman,N.Y., 1992); Lehninger, Biochemistry, Second Edition (Worth Publishers,New York, 1975); Strachan and Read: Human Molecular Genetics, SecondEdition (Wile Liss, N.Y., 1999): Abbas et al, Cellular and MolecularImmunology, 6^(th) edition (Saunders, 2007).

“Aligning” means a method of comparing a test sequence, such as asequence read, to one or more reference sequences to determine whichreference sequence or which portion of a reference sequence is closestbased on some sequence distance measure. An exemplary method of aligningnucleotide sequences is the Smith Waterman algorithm. Distance measuresmay include Hamming distance, Levenshtein distance, or the like.Distance measures may include a component related to the quality valuesof nucleotides of the sequences being compared.

“Amplicon” means the product of a polynucleotide amplification reaction;that is, a clonal population of polynneleotides. Which may be singlestranded or double stranded, which polynucleotides are replicated fromone or more starting sequences. The one or more starting Sequences maybe one or more copies of the same sequence, or they may be a mixture ofdifferent sequences. Ampilcons may be produced by a variety ofamplification reactions whose products comprise replicates of the one ormore starting, or target, nucleic acids. In one aspect, amplificationreactions producing ampilcons are “template-driven” in that base pairingof reactant, either nucleotides or oligonucleotides, have complements ina template polynucleotide that are required, for the creation ofreaction products. In one aspect, template-driven reactions are primerextensions with a nucleic acid polymerase or oligonucleotide ligationswith a nucleic acid ligase. Such reactions include, but are not limitedto, polymerases chain reactions (PCRs), linear polymerases reactions,nucleic acid sequence-based amplification (NASBAs), rolling circleamplifications, and the like, disclosed in the following references thatare incorporated herein by reference: Mullis et al, U.S. Pat. Nos.4,683,195: 4,965,188, 4,683,202; 4,800,159 (PCR): Gelfand et al, U.S.Pat. No. 5,210,015 treat-time PCR with “taqman” probes): Witmer et al,U.S. Pat. No. 6,174,670; Kacian et al, U.S. Pat. No. 5,399,491(“NASBA”); Lizardi,U.S. Pat. No. 5,854,033; Aono et al, Japanese patentpubl. JP 4-262799 (rolling circle amplification); and the like. In oneaspect, amplicons of the invention are produced by PCRs. Anamplification reaction may be a “real-time” amplification if a detectionchemistry is available that permits a reaction product to be measured asthe amplification reaction progresses, e.g. “real-time PCR” describedbelow, or “real-time NASBA” as described in Leone et al, Nucleic AcidsResearch, 26: 2150-2155 (1998), and like references. As used herein, theterm “amplifying” means performing an amplification reaction. A“reaction mixture” means a solution containing all the necessaryreactant for performing a reaction, which may include, but not belimited to, buffering agents to maintain pH at a selected level during areaction, salts, co-factors, scavengers, and the like.

“Clonality” as used herein meads a measure of the degree to which thedistribution of clonotype abundances among clonotypes of a repertoire isskewed to a single or a few clonotypes. Roughly, clonality is an inversemeasure of clonotype diversity. Many measures or statistics areavailable from ecology describing species-abundance relationships thatmay be used for clonality measures in accordance with the inventione,g.. Chapters 17 &, 18, in Pielou. An Introduction to MathematicalEcology, (Wiley-interscience, 1969). In one aspect, a clonality measureused with the invention is a function of a clonotype profile (that is,the number or distinct clonotypes detected and their abundances), sothat after a clonotype profile is measured, clonality may be computedfrom it to give a single number. One clonality measure is Simpson'smeasure, Which is simply the probability that two randomly drawnclonotypes will be the same, Other clonality measures includeinformation-based measures and Mcintosh's diversity index, disclosed inPielou (cited above).

“Clonotype” means a recombined nucleic acid of a lymphocyte whichencodes an immune receptor or a portion thereof. More particularly,clonotype means a recombined nucleic acid, usually extracted from a Tcell or B cell, but which may alson be from a cell-free source, whichencodes a T cell receptor (TCR) or B cell receptor (BCR), or a portionthereof. In various embodiments, clonotypes may encode all or a portionof a VDJ rearrangement of IgH, a DJ rearrangement of IgH, a VJrearrangement of IgK, a VJ rearrangement of IgL, a VDJ rearrangement ofTCR β, a DJ rearrangement of TCR β, a VJ rearrangement of TCR α, a VJrearrangement of TCRγ, a VDJ rearrangement of TCR δ, a VD rearrangementof TCR δ, a Kde-V rearrangement, or the like. Clonotypes may also encodetranslocation breakpoint regions involving immune receptor genes, suchas Bcl1-IgH Bcl1-IgH. In one aspect, clonotypes have sequences that aresufficiently long to represent or reflect the diversity of the immunemolecules that they are derived from consequently, clonotypes may varywidely in length. In some embodiments, clonotypes have lengths in therange of from 25 to 400 nucleotides; in other embodiments, clonotypeshave lengths in the range of from 25 to 200 nucleotides,

“Clonotype profile” means a listing of distinct clonotypes and theirrelative abundances that are derived from a population of lymphocytes,where, for example, relative abundance may be expressed as a frequencyin a given population (that is, a number between 0 and 1). Typically,the population of lymphocytes are obtained from a tissue sample. Theterm “clonotype profile” is related to, but more general than theimmumology concept of immune “repertoire” as described in references,such as the following: Arstila et al, Science, 280: 958-961 (1999):Yassai et al, Immunogenetics, 61: 493-502 (2009): Kedzierska et al, Mol,Immunol., 45(3): 607-618 (2008): and the like. The term “clonotypeprofile” includes a wide variety of lists and abundances of rearrangedimmune receptor-encoding nucleic acids, which may be derived fromselected subsets of lymphocytes (e.g. tissue-infiltrating lymphocytes,immunophenotypic subsets, or the like), or which may encode portions ofimmune receptors that have reduced diversity as compared to full immunereceptors. In some embodiments, clonotype profiles may comprise at least10³ distinct clonotypes; in other embodiments, clonotype profiles maycomprise at least 10⁸ distinct clonotypes: other embodiments, clonotypeprofiles may comprise at least 10⁵ distinct clonotypes; in otherembodiments, clonotype profiles may comprise at least 10⁶ distinctclonotypes. Such embodiments, such clonotype profiles may furthercomprise abundances or relative frequencies of each of the distinctclonotypes. In one aspect, a clonotype profile is a set of distinctrecombined nucleotide sequences (with their abundances) that encode Treceptors (TCRs) or B cell receptors (BCRs), or fragments thereof,respectively, in a population of lymphocytes of an individual, whereinthe nucleotide sequences of the set have a one-to-one correspondencewith distinct lymphocytes or their clonal sub populations forsubstantially all of the lymphocytes of the population. In one aspect,nucleic acid segments defining clonotypes are selected so that theirdiversity (i.e. the number or distinct nucleic acid sequences in theset) is large enough so that substantially ever T cell or B cell orclone thereof in an individual carries a unique nucleic acid sequence ofsuch repertoire. That is, preferably each different clone of a samplehas different clonotype. In other aspects of the invention, thepopulation of lymphocytes corresponding to a repertoire may becirculating B cells, or may be circulating T cells, or may be subpopulations of either of the foregoing populations, including but notlimited to, CD4+ T cells, or CD8+ T cells, or other sub populationsdefined by cell surface, markers, or the like. Such sub populations maybe acquired by taking samples from particular tissues, e.g. bone marrow,or lymPh nodes, or the like, or by sorting or enriching cells from asample (such as peripheral blood) based on one or more cell surfacemarkers, size, morphology, or the like. In still other aspects, thepopulation of lymphocytes corresponding to a repertoire may be derivedfrom disease tissues, such as a tumor tissue, an infected tissue, or thelike. In one embodiment, a clonotype profile comprising human TCR βchains or fragments thereof comprises a number of distinct nucleotidesequences in die range of from 0.1×10⁶ to 1.8×10⁶, or in the range offrom 0.5×10⁴ to 1.5×10⁶, or in the range of from 0.8×10⁶ to 1.2×10⁶. Inanother embodiment, a clonotype profile comprising human IgH chains orfragments thereof comprises a number of distinct nucleotide sequences inthe range of from 0.1×10⁶ to 1.8×10⁶, or in the range of from 0.5×10⁶ to1.5×10⁶, or in the range of from 0.8×10⁶ to 1.2×10⁶. In a particularembodiment, a clonotype profile of the invention comprises a set ofnucleotide sequences encoding substantially all segments of the V(D)Jregion of an chain. In one aspect, “substantially all” as used hereinmeans every segment having a relative abundance of 0.001 percent orhigher; or in another aspect, “substantially all” as used herein meansevery segment having a relative abundance of 0.0001 percent or higher.In another particular embodiment, a clonotype profile of the inventioncomprises a set of nucleotide sequences that encodes substantially allsegments of the V(D)J region of a TCR β chain. In another embodiment, aclonotype profile of the invention comprises a set of nucleotidesequences having lengths in the range of front 25-200 nucleotides andincluding segments oldie V, D, and J regions of a TCR β chain. Inanother embodiment, a clonotype profile of the invention comprises a setof nucleotide sequences having lengths in the range of from 25-200nucleotides and including segments of the V, D, and J regions of an IgHchain. In another embodiment, a clonotype profile of the inventioncomprises a number of distinct nucleotide sequences that issubstantially equivalent to the number of lymphocytes expressing adistinct IgH chain. In another embodiment, a clonotype profile of theinvention comprises a number of distinct nucleotide sequences that issubstantially equivalent to the number of lymphocytes expressing adistinct TCR β chain. In still another embodiment, “substantiallyequivalent” means that with ninety-nine percent probability a clonotypeprofile will include a nucleotide sequence encoding an IgH or TCR β orportion thereof carried or expressed by every lymphocyte of a populationof an individual at a frequency of 0.001 percent or greater. In stillanother embodiment, “substantially equivalent” means that withninety-nine percent probability a repertoire of nucleotide sequenceswill include a nucleotide sequence encoding an IgH or TCR β or portionthereof carried or expressed by every lymphocyte present at a frequencyof 0.0001 percent or greater. In some embodiments, clonotype profilesare derived from samples comprising from 10⁵ to 10⁷ lymphocytes. Suchnumbers of lymphocytes may be obtained from peripheral blood samples offrom 1-10 mL.

“Complementarily determining regions” (CDRs) mean regions of animmunoglobulin (i.e.. antibody) or T cell receptor where the moleculecomplements an antigen's conformation, thereby determining themolecule's specificity and contact with a specific antigen. T cellreceptors and iminunoglobulins each have three CDRs: CDR1 and CDR2 arefound in the variable (V) domain, and CDR3 includessome of V, all ofdiverse (D) (heavy chains only) and joint (J), and some of the constant(C) domains.

“Clonotype Database” means a collection of clonotypes formatted andarranged for ease and speed of searching, comparing and retrieving. Insome embodiments, a clonotype database comprises a collection ofclonotypes encoding the same region or segment of an immune receptor. Insome embodiments, a clonotype database comprises clonotypes of clonotypeprofiles hum a plurality of individuals. In some embodiments, aclonotype database comprises clonotypes of clonotype profiles of atleast 10⁴ clonotypes from at least 10 individuals. In some embodiments,a clonotype database comprises at least 10⁶ in clonotypes, or at least10⁶ clonotypes, or at least 10⁹ clonotypes, or at least 10¹⁰ clonotypes.A clonotype database may be a public database containing clonotypes,such as IMGT database (www.imgt.org), e.g. described in Nucleic AcidsResearch, 31: 307-310 (2003). Clonotype databases may be in a FASTAformat, and clonotype database entries may be searehed or compared usinga BLAST algorithm, e.g..Altschul et al, J. Mol Biol., 215(3): 403-410(1990), or like algorithm.

“Coalescing” means treating two candidate clonotypes with sequencedifferences as the same by determining that such differences are due toexperimental or measurement error and not due to genuine biologicaldifferences. In one aspect, as sequence of a higher frequency candidateclonotype is compared to that of a lower frequency candidate clonotypeand if predetermined criteria are satisfied then the number of lowerfrequency candidate clonotypes is added to that of the higher frequencycandidate clonotype and the lower frequency candidate clonotype isthereafter disregarded. That is, the read counts associated with thelower frequency candidate clonotype are added to those of the higherfrequency candidate clonotype and the higher frequency candidateclonotype and the lower frequency candidate clonotype are treated as thesame that is, the observed difference between them is determined to bedue to error (e.g. Sequencing error, amplification error, or the like).In some embodiments, the predetermined criteria is a likelihood functionthat depends on factors such relative frequencies of the candidateclonotypes being compared, the number of positions at which thecandidates differ, the quality scores of the positions, and the

“Complementarily determining regions” (CDRs) mean regions of animmunoglobulin (i.e., antibody) or T cell receptor where the moleculecomplements an antigen's conformation, thereby determining themolecule's specificity and contact with a specific antigen. T cellreceptors and immunoglobulins each have three CDRs: CDR1 and CDR2 arefound in the variable (V) domain, and CDR3 includes some of V all ofdiverse (D) (heavy chains only) and joint (J), and some of the constant(C) domains.

“Contamination” as used herein means the presence in a tissue sample ofone individual of nucleic acid from another individual. In one aspect,“contamination” means the presence of nucleic acid not originating fronta patient which may affect the interpretation of a clonotype profile ofthe patient.

“Genetic identification” means a unique correspondence between anindividual and a set of values (or states) of genetic markers from oneor more genetic loci of the individual.

“Genetic, marker” mean a polymorphic segment of DNA at a genetic locus,which may be used to identify an individual. A genetic marker may beidentified by its sequence or by adjacent or flanking sequences.Typically, a genetic marker can have a plurality of sequences, orvalues, in different individuals of a population. Exemplary geneticmarkers include, but are not limited to, short tandem repeats (STRs)single nucleotide polymorphisms (SNPs), and the like. The polymorphicsegment of DNA may be genomic DNA or it may be reverse transcribed RNA.In one embodiment, the polymorphic segment is genomic DNA. In oneembodiment, a genetic marker for use with the invention is identified byamplification and sequencing using conventional techniques. In anotherembodiment, genetic markers are amplified and sequenced together withimmune molecules during the process for generating a clonotype profile,

“internal standard” means a nucleic acid sequence that is processed inthe same reaction as one or more target polynucleades in order to permitabsolute or relative quantification of the target polynucleotides in asample. In one aspect the reaction is an amplification reaction, such asPCR. An internal standard may be endogenous or exogenous. That is, aninternal standard may occur naturally in the sample, or it may be addedto the sample prior to a reaction. In one aspect, one or more exogenousinternal standard sequences may be added to a reaction mixture inpredetermined concentrations to provide a calibration to which anamplified sequence may be compared to determine the quantity of itscorresponding target polynucleotide in a sample. Selection of thenumber, sequences, lengths, and other characteristics of exogenousinternal standards is a routine design choice for one of ordinary skillin the art. Endogenous internal standards, also referred to herein as“reference sequences,” are sequences natural to a sample that correspondto minimally regulated genes that exhibit a constant and cellcycle-independent level of transcription, e,g. Selvey et at, Mol. CellProbes, 15: 307-311 (2001), Exemplary internal standards include, butare not limited to, sequences from the following genes: GAPDH,β₂-microglobulin, 18S ribosomal RNA, and β-actin

“Kit” refers to any delivery system for delivering materials or reagentsfor carrying out a method of the invention. In the context of methods ofthe invention, such delivery systems include systems that allow for thestorage, transport, or delivery of reaction reagents (e.g., printers,enzymes, internal standards, etc. In the appropriate containers) and/orsupporting materials (e.g., Wirers, written instructions for performingthe assay etc.) from one location to another. For example, kits includeone or more enclosures boxes) containing the relevant reaction reagentsand/or supporting materialS. Such contents may be delivered to theintended recipient together or separately. For example, a firstcontainer may contain an enzyme for use in an assay, while a secondcontainer contains primers.

“Minimal residual disease” means remaining cancer cells after treatment.The term is frequently used in connection with treatment of lymphomasand leukemias.

“Lymphoid or myeloid proliferative disorder” means any abnormalproliferative disorder in which one or more nucleotide sequencesencoding one or more rearranged immune receptors can be used as a markerfor monitoring such disorder. “Lymphoid or myeloid neoplasm” means anabnormal proliferation of lymphocytes or meloid cells that may bemalignant or non,malignant. A lymphoid cancer is a malignant lymphoidneoplasm. A myeloid cancer is a malignant myeloid neoplasm. Lymphoid andmyeloid neoplasms itre the result of or are associated with,lymphoproliferative or myeloproliferative disorders, and include, butare not limited to, follicular lymphoma, chrome lymphocyte leukemia(CLL), acute lymphocyte leukemia (ALL), chronic myelogenous leukemia(CML), acute myelogenous leukemia (AML), Hodgkins's and non-Hodgkin'slymphomas, multiple mytiloina (MM), monoclonal gammopathy ofundeterinined significance (MGUS), mantle cell lymphoma (MCL), diffuselarge B cell lymphoma (DLBCL), myelodysplastic. syndromes (MDS). T celllymphoma, or the like, e.g. Jaffe et al, Blood, 112: 4384-4399 (2008);Swerdlow et al, WHO Classification of Tumours of Haernatopoietic andLymphoid Tissues (e, 4^(th)) (IARC Press, 2008), As used herein, “B cellcancer” means a lymphoid or myeloid neoplasm involving B cells or celladeveloped therefrom, such as plasma cells. Likewise, “T cell cancer”means a lymphoid or myeloid neoplasm involving T cells or cellsdeveloped therefrom.

“Percent homologous,” “percent identical,” or like terms used inreference to the comparison of a reference sequence and another sequence(“comparison sequence”) mean that in an optimal alignment between thetwo sequences, the comparison sequence is identical to the rekrencesequence in a number of subunit positions equivalent to the indicatedpercentage, the subunits being nucleotides for polynucleotidecomparisons or amino acids for polypeptide comparisons. As used herein,an “optimal alignment” of sequences being compared is one that maximizesmatches between subunits and minimizes the number of gaps employed inconstructing an alignment. Percent identities may be determined withcommercially available implementations of algorithms, such as thatdescribed by Needleman and Wunsch, J. Mol. Bioil., 48: 443-453(1970)(“GAP” program of Wisconsin Sequence Analysis Package, GeneticsComputer Group, Madison, Wis.), or the like. Other software packages inthe art for constructing alignments and calculating percentage identityor other measures of similarity include the “BestFit” program, based onthe algorithm of Smith and Waterman, Advances in Applied Mathematics, 2:482-489 (1981) (Wisconsin Sequence Analysis Package, Genetics ComputerGroup, Madison, Wis.). In other words, for example. to obtain apolynucleotide having a nucleotide sequence at least 95 percentidentical to a reference nucleotide sequence, up to five percent of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide. or a number of nucleotides up to live percent of thetotal number of nucleotides in the reference sequence may be insertedinto the reference sequence.

“Polymerase chain reaction,” or “PCR,” means a reaction for the in vitroamplification of specific DNA sequences by the simultaneous primerextension of complementary strands of DNA. In other words, PCR is areaction for making multiple. copies or replicates of a target nucleicacid flanked by primer binding sites, such reaction comprising one ormore repetitions or the following steps: (i) denaturing the targetnucleic acid, (ii) annealing primers to the primer binding sites. and(iii) extending the primers by a nucleic acid polyinerase in thepresence or nucleoside niphosphates. As used herein. the terms “forwardprimer” and “upstream primer” are used interchangeably, and the terms“reverse primer” and “downstream primer” are used interchangeably. Alsoas used herein, if a double stranded target polynucleotide is displayedwith its sense strand in a 5′→3′ left-to-right orientation, a forwardprimer would bind to the antisense strand on the left and be extended tothe right and a reverse primer would bind to the sense strand on theright and be extended to the left. Usually. the reaction is cycledthrough different temperatures optimized for each step in a thermalcycler instrument. Particular temperatures, durations at each step, andrates of change between steps depend on many factors well-known to thoseof ordinary skill in the art, e.g., exemplified by the references:McPherson et al, editors, PCR: A Practical Approach and PCR2: APractical Approach (IRL Press, Oxford, 1991 and 1995, respectively). Forexample, in a conventional PCR using Taq DNA polymerase, a doublestranded target nucleic acid may be denatured at a temperature >90° C.,primers annealed at a temperature in the range 50-75° C., and primersextended at a temperature in the range 72-78° C. The term “TCR”encompasses derivative forms of the reaction, including but not limitedto, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexedPCR, and the like. Reaction volumes range from a few hundred nanoliters,e.g. 200 nL, to a few hundred μL, e.g., 200 μL. “Reverse transcriptionPCR,” or “RT-PCR,” means a PCR that is preceded by a reversetranscription reaction that converts a target RNA to a complementarysingle stranded DNA, which is then amplified, e.g., Tecott et. al, U.S.Pat. No. 5,168,038, which patent is incorporated herein by reference.“Real-time PCR” means a PCR for which the amount of reaction product,i.e, amplicon, is monitored as the reaction proceeds. There are manyforms of real-time PCR that differ mainly in the detection chemistriesused for monitoring the reaction product, e.g. Gelfand et al, U.S. Pat.No. 5,210,015 (“taqman”); Witwer et al, U.S. Pat. Nos. 6,174,670 and6,569,627 (intercalating dyes); Tvagi et al, U.S. Pat. No. 5,925,517(molecular beacons); which patents are incotporatcd herein by reference.Detection chemistries for real-time PCR are reviewed in Mackay et al,Nucleic Acids Research, 30: 1292-1305 (2002), which is also incorporatedherein by reference. “Nested PCR” means a two-stage PCR wherein theamplicon of a first PCR becomes the sample for a second PCR using a newset of primers, at least one of which binds to an interior location ofthe first amplicon. As used herein, “initial primers” in reference to anested amplification reaction mean the primers used to generate a firstamplicon, and “secondary primers” mean the one or more primers used togenerate a second, or nested, amplicon. “Multiplexed PCR” means a PCRwherein multiple target sequences (or a single target sequence and oneor more reference sequences) are simultaneously carried out in the samereaction mixture, e.g. Bernard et al, Anal. Biochem., 273: 221-228(1999)(two-color real-time PCR). Usually, distinct sets of primers areemployed for each sequence being amplified. “Quantitative PCR” means aPCR designed to measure the abundance of one or more specific targetsequences in a sample or specimen. Quantitative PCR includes bothabsolute gnawnation and relative quantitation of such target sequences.Quantitative measurements are made using one or more reference sequencesor internal standards that may be assayed separately or together with atarget sequence. The reference sequence may be endogenous or exogenousto a sample or specimen, and in the latter case, may comprise one ormore competitor templates. Typical endogenous reference sequencesinclude segments of transcripts of the following genes: β-actin, GAPDH,β₂-microglobutin, ribosomal RNA, and the like. Techniques forquantitative PCR are well-known to those of ordinary skill in the art,as exemplified in the following references that are incorporated byreference: Freeman et al, Biotechniques, 26: 112-126 (1999);Becker-Andre et al, Nucleic Acids Research, 17: 9437-9447 (1989);Zimmerman et al, Biotechniques, 21: 268-279 (1996); Diviacco et al..Gene, 122: 3013-3020 (1992); Becker-Andre et al, Nucleic Acids Research,17: 9437-9446 (1989); and the like.

“Primer” means an ofigoilucleotide, either natural or synthetic that iscapable, upon forming a duplex with a polynuclemide template, of actingas a point of initiation of nucleic acid synthesis and being extendedfrom its 3′ end along the template so that an extended dnplex is formed.Extension of a primer is usually carried out with a nucleic acidpolymerase, such as a DNA or RNA polymerase. The sequence of nucleotidesadded in the extension process is determined by the sequence of thetemplate polynucleotide, Usually primers are extended by a DNApolymerase. Primers usually have a length in the range of fmin 14 to 40nuclexltides, or in the rang,e of from 18 to 36 nucleotides. Primers areemployed in a variety of nucleic amplification reactions.. for example,linear amplification reactions using a single primer, polymerase chainreactions, employing two or more primers. Guidance for selecting thelengths and sequences of primers for particular applications is wellknown to those of ordinary skill in the art, as evidenced by thefollowing references that are incorporated by reference: Dieffenbach,editor, PCR Primer: A Laboratory Manual, 2^(nd) Edition (Cold SpringHarbor Press, New York, 2003).

“Quality score” means a measure of the probability that a baseassignment at a particular sequence location is correct, A varietymethods are well known to those of ordinary skill kir calculatingquality scores for particular circumstances, such as, thr bases calledas a result of different sequencing chemistries, detection systems,base-carling algorithms, and so on. Generally, quality score values aremonotonically related to probabilities of correct base calling. Forexample, a quality score, or Q, of 10 may mean that there is a 90percent chance that a base is called correctly, a Q of 20 may mean thatthere is a 99 percent chance that a base is called correctly, and so on.For some sequencing platforms, particularly those usingsequencing-by-synthesis chemistries, a vuage. quality scores decrease asa function of wquence read length, so that quality scores at thebeginning of a sequence read are higher than those at the end of asequence read, such declines being due to phenomena such as incompleteextensions, carry forward extensions, loss of template, loss ofpolymerase, capping failures, deprotection failures, and the like.

“Sequence read” means a sequence or nucleotides determined from asequence or stream of data generated by a sequencing technique, whichdetermination is made, for example, by means of base-calling softwareassociated with the technique, e.g. base-caning software from acommercial provider of a DNA sequencing: platform. A sequence readusually includes quality scores for each nuctemide irt the sequence.Typically, sequence reads are made by extending a primer along atemplate nucleic acid, e,g, with a DNA polymerase or a DNA ligase Datais generated by recording signals, such as optical, chemical (e.g. pHchange), or electrical signals, associated with such extension. Suchinitial data is converted into a sequence read.

“Sequence tag” (or “tag”) “bareode” means oligonucleotide that isattached to a polynucleotide or template molecule and is used to .identiand/or track the polynucleotide or template in a reaction or a series ofreactions. Each sequence tag has a nucleotide sequence which issometimes referred to herein as a “tag sequence.” A sequence tag may beattached to the 3′-5′-end of a polynucleotide or template or it may beinserted into the interior of such polynucleotide or template to form alinear or circular conjugate, sometime referred to herein as a “taggedpolynucleotidc,” or “tagged template,” or “tag-polyancleotideconjugate,” “tag-molecule conjugate,” or the like, Sequence tags mayvary widely in size and compositions', the following references, whichare incorporated herein by reference, provide guidance for selectingsets of sequence tags appropriate for particular embodiments: Brenner,U.S. Pat. No. 5,635,400; Brenner and Macevicz, U.S. Pat. No. 7,537,897:Brenner et al, Proc. Natl. Acad. Sci., 97: 1605-1670 (2000); Church etal, European patent publication 0 303 459; Shoemaker et al, NatureGenetics, 14: 450-456(1996); Morris et al, European patent publication0799897A1; Wallace, U.S. Pat. No. 5,981,179; and the like. Selection ofparticular tag lengths and/or compositions my depend on several factorsincluding, without limitation, the sequencing technology used to decodea tag, the number of distinguishable tags required to unambigmouslyidentify a set of target polynucleotides, how different must rags of aset be in order to ensure reliable identification, e.g. freedom fromcross hybridization or misidentification from sequencing errors, and thelike. In some embodiments, sequence tags can each have a length within arange of from 6 to 100 nucleotides, or from 10 to 100 nucleotides, orfrom 12 to 50 nucleotides, or from 12 to 25 nucleotides, respectively.In some embodiments, sets of sequence tags are used wherein eachsequence tag of a set has a unique nucleotide sequence that differs fromthat of every other tag of the same set by at least four bases; in otherembodiments, sets of sequence tags are used wherein the sequence of eachtag of a set differs from that of every other tag of the same set by at.least five bases; in still other embodiments, sets or sequence tags areused wherent the sequence of each tag of a set differs from that ofevery other tag of the same set by at least ten percent of theirnucleotides; or in other embodiments, at least twenty-five percent oftheir nucleotides; or in other enthodiments, at least fifty percent oftheir nucleotides.

What is claimed is:
 1. A method of generating clonotype profiles from aplurality of Mutt le cell receptor chains, the method comprising thesteps of: (a) combining in a reaction mixture under primer extensionconditions a first set of primers with a sample of recombined nucleicacids from B-cells and/or T-cells and/or cell-free DNA, wherein eachprinter of the first set has a receptor-specific portion such that thereceptor-specific portion anneals to a different recombined nucleic acidat a predetermined location and is extended to form a first extensionproduct, and wherein each primer of the first set has a5′non-eomplementary end containing a first primer binding site (b)removing from the reaction mixture non-extended primers of the firstset; (c) adding to the reaction mixture under primer extensionconditions a second set of primers, wherein each primer of the secondset has a receptor-specific portion such that the receptor-specificportion anneals to the first extension product at a predeterminedlocation and has a 5′-non-complementary end containing a second primerbinding site, primers of the first set and/or primers of the second setcomprising a sequence tag disposed between the receptor-specific portionand the first or second primer binding site, respectively, and whereineach primer of the second set is extended to form a second extensionproduct, such that each second extension product comprises a firstprimer binding site, a second primer binding site, at least one sequencetag, and recombined nucleic acid encoding a portion or T cell receptorchain or a B cell receptor chain; (d) performing a polymerase chainreaction in the reaction mixture to form an amplicon, the polymerasechain reaction using forward primers specific for the first primerbinding site and reverse primers specific for the second primer bindingsite; and (e) sequencing the nucleic acids or the amplicon to form aclonotype profile of a plurality of T cell receptor chains and/or B cellreceptor chains.
 2. The method of claim 1 wherein said plurality of Tcell receptor chains and/or B cell receptor chains comprises TCRβ, TCRδand TCRγ and wherein said primers of said first set and said primer ofsaid second set comprise primers that flank regions of said recombinednucleic acids encoding VDJ regions of TCRβ and TCRδ and primers thatflank VJ regions of TCRγ.
 3. The method of claim 1 wherein said step ofsequencing includes: generating sequence reads each having an error rateand each comprising a tag sequence and a recombined nucleic acidsequence; aligning sequence reads having like tag sequences to formgroups of sequence rends having the same sequence tags; coalescingsequence reads of groups to determine clonotypes, wherein. groups ofsequence reads are coalesced into different recombined nucleic acidsequences whenever said groups of semtence reads are distinct with alikelihood of at least ninety-five percent; and forming said clonotypeprofile from the clonotypes.
 4. The method of claim 1 further includinga.second:step of removing after said second extension product is
 5. Themethod of claim 1 wherein said annealing and extension of said primersof said first set is repeated after melting said first extensionproduct.
 6. The method of claim 1 wherein said annealing and extensionof said primers of said second set is repeated after melting said secondextension product.
 7. The method of claim 1 wherein said step ofremoving includes adding an exonuclease I activity to said reactionmixture.
 8. A method of generating clonotype profiles from a pluralityof B cell receptor chains, the method comprising the steps of: (a)combining in a reaction mixture tinder primer extension conditions afirst set of primers with a sample of recombined nucleic acids fromB-cells and/or cell-free DNA, wherein each primer of the first set has areceptor-specific portion such that the receptor-specific portionanneals to a different recombined nucleic acid at a predeterminedtocition and is extended to form a lust extension product, and where illeach pt nn of the first set has a 5′-non-complementary end containing itfirst primer binding site (b) removing from the reaction mixturenon-extended primers of the first set; (c) adding to the reactionmixture under printer extension conditions a second set of primers,wherein each primer of the second sel has a receptor-specific put donsuch. that the .receptor-pecific portion anneals to the first extensionproduct at a predetermined location and has a 5′-non-complementary endcontaining is second primer binding site, primers of the first setand/or primers of the second set. comprising a sequence tag disposedbetween the receptor-specific portion and the first or second primerbinding site, respectively, and wherein each primer of the second set isextended to form a second extension product, such that each secondextension product comprises a first prhner binding site a seeond primerbinding site, at least one sequence tag, and recombined nucleic acidencoding a portion of a B cell receptor chain; (d) performing apolymerase chain reaction in the reaction mixture to form an amplicon,the polymerase chain reaction using forward primers specific for thefirst primer binding site and reverse primers specific for the secondprinter binding site and (e) sequencing the nucleic acids of theamplicon to farm a clonotype profile of a plurality of B cell receptorchains.
 9. The method of claim 8 wherein said plurality of B cellreceptor chains comprises IgH and IgK and wherein said primers of saidfirst set and said primers of said second set comprise primers thatflank regions of said recombined nucleic xids encoding VDS regions ofIGH, DJ regions of IgH and VJ regions of IgK.
 10. The method of claim 9wherein said primers of said first set or said second set include atleast one nested set of primers specific fir a plurality of differentprimer binding sites in V regions of said inH chains.
 11. The method ofclaim t vherein said step of sequencing includes: generating sequencereads each haying an error rate and each comprising a lag sequence and arecombined nucleic acid sequence; aligning sequence reads haying liketag sequences to form groups of sequence reads having the same sequencetags; coalescing sequence reads of groups to determine clonotypes,wherein groups of sequence reads are coalesced into different recombinednucleic acid sequences whenever said groups of sequence reads aredistract with a likelihood of at least ninety-five percent; and formingsaid clonotype profile from the clonotypes.
 12. The method of claim 8further including a second step of removing after said second extensionproduct is fonned.
 13. The method of claim 8 wherein said annealing andextension of said primers of said first set is repeated after melting,said first extension product.
 14. The method of claim 8 wherein saidannealing and extension of said primers of said second set is repeatedafter melting said second extension product.
 15. The method of claim 8wherein said step of removing includes adding an exorinclease I activityto said reaction mixture.
 16. A method of determining a clonotypeprofile of recombined nucleic acids encoding a plurality of immunereceptor chains in a sample, the method comprising the steps of: (a)obtaining a sample from an individual comprising T-cells and/or B-cellsand/or cell-free DNA; (b) attaching sequence tags to recombined nucleicacid molecules of T-cell receptor genes or immunoglobulin genes from thesample to form tag-nucleic acid conjugates, wherein at least onerecombined nucleic acid or copies thereof:have different sequence tagsattached; (c) amplifying the tag-nucleic acid conjugates; (d)sequencing. a sample of the tag iucleic acid coniugates to providesequence reads each having an error rate and each comprising a tagsequence and a recombined nucleic acid sequence; (e) aligning sequencereads having like tag sequences to form groups of sequence reads havingthe same sequence tags; (f) coalescing sequence reads of groups todetermine clonotypes, wherein groups of sequence reads are coalescedinto different recombined .nucleic acid sequences whenever said groupsof sequence reads are distinct with a likelihood of at least ninety-fivepercent; and (g) determining the clonotype profile of the sample bydetermining levels of the clonotypes.
 17. The method of claim 16 whereinsaid steps of attaching and amplifying comprise: combining in a reactionmixture under primer extension conditions a first set of primers withsaid sample, wherein each primer of the first set has areceptor-specific portion such that the receptor-specific portionanneals to a different recombined nucleic acid at a predeterminedlocation and is extended to form a first extension product, and whereineach primer of the first set has a 5′-non-complementary end containing afirst primer binding site; removing from the reaction mixture nonextended primers of the first set; adding to the reaction mixture underprimer extension conditions a second set o primers, wherein each primerof the second set has a :receptor-specific portion such that thereceptor-specific portion anneals to the first extension product at apredetermined location and has a 5′-non-complementary end containing asecond primer binding site, primers of the first set and/or primers ofthe second set comprising a sequence tag disposed between thereceptor-specific portion and the first or second primer binding site,respectively, and wherein each primer of the second set is extended toform a second extension product, such that each second extension productcomprises a first primer binding site, a second primer binding site, atleast one sequence tag, and recombined nucleic acid encoding a portionof a T cell receptor chain or a B cell receptor chain; and performing apolymerase chain reaction in the reaction mixture to form an amplicon,the polymerase chain reaction using forward primers specific for thefirst primer binding site and reverse primers specific for the secondpriater binding site.
 18. The method of claim 17 further including asecond step of removing after said second extension product is formed.19. The method of claim 17 wherein said annealing and extension of saidprimers of said first set is repeated after melting said first extensionproduct.
 20. The method of claim 17 wherein said annealing and extensionof said primers of said second set is repeated after melting said secondextension product.
 21. The method of claim 17 wherein said step ofremoving includes adding an exonuclease I activity to said reactionmixture.
 22. The method of claim 17 wherein said steps of attaching andamplifying generate tag-nucleic acid conjugates wherein at least onesaid recombined nucleic acid and its respective copies have a pluralityof different sequence tags attached.
 23. The method of claim 22 whereinsaid recombined nucleic, acids comprise recombined nucleic acidsencoding TCRβ, TCRδ and TCRγ chains and wherein said primers of saidfirst set and said primer of said second set comprise primers that flankregions of said recombined nucleic acids encoding VDJ regions of TCRβand TCRδ and primers that flank VJ regions of TCRγ.
 24. The method ofclaim 22 wherein said recombined nucleic acids comprise recombinednucleic a ids encoding IgH and IgK and wherein said primers of saidfirst set and said primers of said second set comprise primers thatflank regions of said recombined nucleic acids encoding VDJ regions ofIgH, DJ regions of IgH and VJ regions of IgK.
 25. The method of claim 16wherein said step of coalescing includes coalescing said groups ofsequence reads into different recombined nucleic acid sequences wheneversaid groups of sequence reads are distinct with a likelihood of at least99.9 percent.
 26. A method of monitoring a minimal residual disease of alymphoid proliferative disorder, the method comprising the steps of: (a)obtaning from an individual a sample containing T-cells and/or B-cellsand/or cell free DNA or RNA; (b) attaching sequence tags to each of aplurality of recombined nucleic acids in the sample to form tag-nucleicacid conjugates, wherein at least one recombined nucleic acid or copiesthereof have different sequence tags attached and are characteristic ofa lymphoid proliferative disorder of the individual; (c) amplifying thetag-nucleic acid conjugates; (d) sequencing a sample of the tag-nucleicacid conjugates to provide sequence reads each comprising a tag sequenceand a recombined nucleic acid sequence; (e) aligning sequence readshaving like tag squences to form groups of sequence reads having thesame sequence tags; (f) coalescing recombined nucleic acid seqtiences ofgroups to deteremine clonoltypes, wherein groups of sequence reads arecoalesced into different clonotypes whenever said groups of recombinednucleic acid sequences are distinct with a likelihood of at least 99.9percent; and (g) detecting in a clonotype profile the presence, absenceand/or level of clonotypes correlated with the lymphoid proliferativedisorder of the individual.
 27. The method of claim 26 wherein saidsteps of attaching, and amplifying comprise: combining in a reactionmixture under primer extension conditions a first set of primers with asample of recombined nucleic acids from B-cells and/or and/or cell-freeDNA, wherein each primer of the first set has a receptor-specificportion such that the receptor-specific portion anneals to a differentrecombined nucleic acid at a predetermined location and is extended toform a first extension product, and wherein each primer of the first sethas a 5′-non-complementary end containing a first primer binding site;removing from the reaction mixture non extended primers of the firstset; adding to the reaetion mixture under primer extension conditions asecond set of primers, wherein each primer of the second set has areceptor-specific portion such that the receptor-specific portionanneals to the first extension product at a predetermined location andhas a 5′-non-complementary end containing a second primer binding site,primers of the first set and/or primers of the second set comprising asequence tag disposed between the receptor-specific portion and thefirst or second primer binding site, respectively, and wherein eachprimer of the second set is extended to form a second extension product,such that each second extension product comprises a first primer bindingsite, a second primer binding site, at least one sequence tag, andrecombined nucick acid encodim a portion or a T cell receptor chain or aB cell receptor chain; performing a polymerase chain reaction in thereaction mixture to form an amplicon, the polymerase chain, reactionusing forward primers specific for the first primer binding site andreverse primers specific for the second primer binding site.
 28. Themethod of claim 27 wherein said recombined nucleic acids compriserecombined nucleic acids encoding TCRβ, TCRδ and TCRγ chains and whereinsaid primers of said first set and said primer of said second setcomprise primers that flank regions of said recombined nucleic acidsencoding VDJ regions of TCRβ and TCRδ and primers that flank VJ regionsof TCRγ.
 29. The method of claim 27 wherein said recombined nucleicacids comprise recombined nucleic acids encoding IgH and IgK and whereinsaid primers of said first set and said printers of said second setcomprise primers that flank regions of said recombined nucleic acidsencoding VDJ regions of IgH, DJ regions of IgH and VJ regions of IgK.30. The method of claim 29 wherein said primers of said first set orsaid second set include at least one nested set of primers specific fora plurality of different primer binding sites in V regions of said IgHchains.
 31. The method of claim 30 further including a second step ofremoving after said second extension product is formed.
 32. The methodof claim 30 wherein said annealing and extension of said primers of saidfirst set is repeated after melting said first extension product. 33.The method of claim 3 wherein said anneaiing and extension of saidprimers of said second set is repeated after melting said secondextension product.